-NRLF 


Edmund  O'Neill 


Ocular  or  Eye-piece 


Draw-Tube 


Rack  and  Pinion  for 
coarse  adjustment 


Micrometer 
Screw  for 
fine  adjustment 


Triple  Nose-piece 
Objectives 


Stage 


Abbe  Condenser 
Iris  Diaphragm 

Pinion  for 
oblique  light 


Plane  and 
Concave  Mirror. 


LABORATORY    WORK 


IN 


BACTERIOLOGY 


BY 
FREDERICK  G.   NOW,   Sc.D.,   M.D., 

JUNIOR     PROFESSOR    OF    HYGIENE    AND    PHYSIOLOGICAL    CHEMISTRY, 
UNIVERSITY    OF    MICHIGAN. 


SECOND    EDITION,   REVISED   AND    ENLARGED, 
WITH    FRONTISPIECE    AND    SEVENTY-SIX    ILLUSTRATIONS. 


ANN  ARBOR: 

GEORGE  WAHR,  PUBLISHER. 

1899. 


Copyright,  iSgg,  by  F.  G.  Novy. 


IN  liEMORIAM 


PREFACE. 


A  thorough  course  of  laboratory  instruction  in  bacter- 
iology is  absolutely  essential  to  the  proper  education  of  the 
medical  student  of  the  present  day.  The  practical  knowl- 
edge thus  acquired  in  the  methods  of  handling  bacteria,  in 
the  precautions  necessary  to  the  prevention  of  personal 
infection,  and  in  the  methods  for  the  recognition  and  for 
the  destruction  of  disease-producing  organisms  is  funda- 
mental and  invaluable.  Such  information  is  directly  useful 
as  a  means  of  diagnosis;  it  is  necessary  to  the  successful 
performance  of  antiseptic  operations  and  is  indispensable 
to  the  proper  execution  and  understanding  of  the  common 
hygienic  measures  for  the  prevention  of  communicable 
diseases. 

It  is  therefore  evident  that  the  course  in  bacteriology 
should  not  be  inferior,  either  in  length  or  in  the  character  of 
the  instruction,  to  any  other  laboratory  course  offered  in 
the  medical  curriculum*  The  student  should  be .  taught  to 
work,  not  merely  with  a  few  harmless  bacteria,  but  espe- 
cially with  all  >of  the  common  pathogenic  organisms.  The 
exclusion  of  the  latter  organisms  from  a  laboratory  course 
on  the  plea-- of  danger  is  'an  admission  of  weakness  in 
instruction  or  in  supervision.  '"?•  The  danger  in  a  laboratory  is 
avoidable,  and  is  not  to  be  compared  with  that  encountered 


4  PREFACE. 

in  practice  in  connection  with  cases  of  actual  disease. 
Accidents  may  occur,  it  is  true,  but  they  are  extremely  rare 
as  compared  with  the  numerous  instances  of  infection 
incurred  in  post-mortems  or  in  surgical  operations. 

Bacteriology,  as  an  educational  measure  of  the  first 
importance,  belongs  in  the  first,  or  at  the  latest,  in  the 
second  year  of  a  medical  course.  The  student  is  thus 
enabled  to  make  use  of  his  knowledge  in  connection  with 
his  clinical  studies.  The  spirit  of  scientific  investigation, 
and  not  mere  book  reading,  must  be  fostered  in  the  student 
from  the  outstart,  since  it  is  this  that  leads  to  progress  in 
medicine  and  serves  to  distinguish  the  true  physician  from 
those  bound  down  through  blind  faith,  commercialism  or 
ignorance. 

This  edition  is  thoroughly  revised  and  greatly  enlarged. 
It  should  be  noted  that  it  is  a  text-book  of  laboratory  work 
for  students.  The  arrangement  of  the  subject  matter  is 
different  from  that  usually  met  with  in  text-books,  for  the 
reason  that  it  conforms  as  much  as  possible  to  the  actual 
work  as  carried  on,  day  by  day,  in  the  Hygienic  Laboratory 
of  the  University  of  Michigan.  During  the  past  ten  years 
three  laboratory  courses  in  bacteriology  have  been  given 
annually.  Each  course  covers  a  period  of  twelve  weeks  of 
daily  work,  to  which  the  entire  afternoon  is  devoted.  Inas- 
much as  this  laboratory  work  is  required  of  all  medical 
•students,  the  number  of  students  which  annually  take  the 
course  at  times  exceeds  two  hundred. 

Since  it  is  a  beginner's  guide  and  not  a  manual,  it 
obviously  is  undesirable  to  introduce  the  numerous  modifi- 


PREFACE.  5 

cations  that  can  be  found  of  almost  every  known  procedure. 
The  methods  that  are  described  are  those  which  have  stood 
the  severe  test  of  the  practical  instruction  indicated  above. 
Many  of  the  methods,  as  well  as  some  of  the  apparatus 
described,  have  originated  in  this  laboratory.  Illustrations 
of  the  various  bacteria  and  of  their  cultural  characteristics 
have  been  expressly  omitted,  inasmuch  as  the  student  is 
expected  to  sketch  from  observation  the  form  of  each 
organism  and  its  peculiarities  of  growth  in  the  colony,  and 
in  tube  culture.  Blank  pages  are  provided  for  this  purpose 
and  for  such  additional  notes  as  may  be  desirable. 

More  space  has  not  been  given  to  the  consideration  of 
the  individual  pathogenic  bacteria,  and  of  questions  of 
immunity,  for  the  reason  that  these  and  allied  subjects  are 
treated  of  in  a  general  course  of  lectures  which  is  wholly 
independent  from  the  laboratory  work.  These  lectures  on 
general  bacteriology  are  given  daily  and  extend  throughout 
a  semester.  From  the  standpoint  of  a  Jteacher  it  is  desir- 
able that  the  two  courses  be  kept  distinct,  and  for  that 
reason  the  treatment  of  the  general  subject  should  be  pub- 
lished separately,  if  at  all.  An  exception  is  made  in  the 
case  of  the  first  five  chapters,  which  deal  with  the  general 
properties  of  bacteria,  and  which,  as  such,  are  necessary 
for  reading  and  reference  in  connection  with  the  laboratory 
work. 

The  last  two  chapters  are  devoted  to  special  methods 
which  will  be  of  value  to  advanced  students.  Several  of 
the  characteristic  ingenious  methods  of  the  Pasteur  school 
are  given  at  length.  Here,  as  elsewhere  in  the  book,  the 


6  PREFACE. 

attempt  is  made  to  supply  the  fullest  details  to  the  student 
in  order  to  insure  satisfactory  results.  A  complete  index 
will  serve  to  render  the  text  easily  accessible. 

It  is  hardly  necessary  to  state  that  the  various  journals 
and  the  standard  works  on  bacteriology  have  been  freely 
drawn  upon  in  the  preparation  and  revision  of  these  pages. 
It  has  been  deemed  desirable  to  introduce  .illustrations 
especially  of  such  apparatus  as  is  actually  employed* 
Thanks  are  due  to  the  Bausch  &  Lomb  Optical  Co.,  of 
Rochester,  N.  Y. ,  also  to  Eimer  &  Amend,  and  Stransky  & 
Co.,  of  New  York,  for  kindly  supplying-  several  of  the  cuts. 

F.  G.  NOVY. 

ANN  ARBOR.  MICH.,  April  15,  1899. 


CONTENTS 


CHAPTER    I. 

Page. 
Form  and  Classification  of  Bacteria     ....       15 

Definition  of  bacteria. Microscopic  plants  and  animals, 

distinction. Bacteria  as  plants. Bacteria,  fission  fungi  or 

schizomycetes;  moulds,  thread  fungi  or  hyphomycetes;  yeasts, 
budding-  fungi  or  blastomycetes. Absence  of  a  natural  classi- 
fication.  Bacteria  classified  according  to  form;  micrococcus, 

bacillus,   spirillum. Modifications;   bacterium,  vibrio,   spiro- 

chaete. Division  into  species. Influence  of  environment  on 

form  and  size. Involution  forms. Variations  due  to  meth- 
ods of  examination. Plasmolytic  changes. Pleomorphism. 

Constancy  of  species. Attenuation. Origin  of  new  spe- 
cies. 

CHAPTER  II. 

Size  and  Structure  of  the  Bacterial  Cell       '.          .         .       24 

Bacteria  as  unicellular  organisms. Called  the  smallest 

of  living  beings. Existence  of  still  more  minute  life. 

Micromillimeter  or  micron. Size  of  bacteria. 

The  cell-wall,  composition,  demonstration. Plasmolysis. 

Softening  of  the  outer  layer,  the  capsule. Zooglea. 

The  contents  of  the  cell,  composition. Existence  of  a 

nucleus. Appearance  of  contents;  granulations,  polar  bodies, 

color,  absence  of  chlorophyll. Granulose  reaction. 

Motility. Molecular  or  Brownian  movement. Real 

motion,  flagella  or  whips. Number  and  arrangement. 

Giant  whips. 

CHAPTER  III. 

The  Life-history  of  Bacteria        .          .          .        V        .       41 

Rapidity  of  multiplication. Cell  division:  diplo-bacillus, 

threads;  diplococcus,  streptococcus,  tetrad,  sarcine,  staphylo- 
coccus;  vibrio,  spirillum. 


8  CONTENTS. 

Spores. Vegetating-  and  reproductive  forms. Endos- 

pore,   arthrospore. Sporulation. Position    of    the    spore; 

median,  terminal,  intermediate. Resultant  cell  forms;  clos- 

tridium,  "drum-sticks." Cause  of  spore  formation. Aspor- 

ogenic    bacteria. Germination. Spore    structure. Re- 
sistance of  spores. Spontaneous  generation. 


CHAPTER  IV. 

The  Environment  of  Bacteria      .          .          .          .          .58 

Conditions  of  .growth Necessity  of  moisture. Chem- 
ical composition  of  the  cell. Sources  of  carbon,  nitrogen, 

hydrogen,   oxygen,   and    other    elements. Reaction   of    the 

medium. Distribution  of  bacteria  in  nature,  where  absent. 

Classification    according    to  habitat. Saprophytic  and 

parasitic  bacteria. Obligative   and    facultative    forms. — 

Classification  according  to  oxygen    requirements. Aerobic 

and  anaerobic  bacteria. Obligative  and  facultative  forms. 

Microbic  association. 

Temperature,  minimum,  maximum  and  optimum. Ther- 

mophilic  bacteria. Effect  of  cold  and  heat  on  vitality. — 

Action  of  light,  high  pressure,  electricity, Chemotaxis. 

CHAPTER  V. 

The  Chemistry  of  Bacteria 79 

The  number  and  kind  of  products  vary  with  each  species. 
Influence  of  environment. Accumulation  of  waste-pro- 
ducts.— —Synthetic  and  analytic,  primary  and  secondary  pro- 
ducts. 

Bacterial  proteins,  tox-albumins.  — Venoms,  plant  albu- 
moses  as  abrin  and  ricin. Toxins;  synthetic  products,  elabor- 
ated within  the  cell,  not  bases  or  proteins. — -Ferments, 

organized  and  unorganized. Enzymes,  their  classification. 

—  Ptomai'ns. Alkalis. Acids. Alcohols. Gases. 

Classification  of  bacteria  according  to  function. 

Fermentations. Their  cause,  the  nature  of  the  chemical 

changes  induced. Diverse  fermentations,  alcoholic;  acetic 

acid,  vinegar,  summer  complaint;  lactic  acid,  dental  caries, 
stomach  and  intestinal  disorders,  souring  of  milk,  koumiss, 
cheese;  butyric  acid,  sauerkraut,  ensilage,  cheese,  retting; 

viscous  or  slimy  fermentations. Fermentations  of  fats,  flavor 

of  butter. Hydrogen  sulphide  and  ammoniacal  fermentations 

of  urine. Nitrification  in  water  and  soil,  saltpeter. Deni- 

trification. Indigo  reduction. Putrefaction,  bacteria  as 

scavengers  perpetuate  life. 


CONTENTS. 


Pigment  production. Phosphorescence. Heat  produc- 
tion.  Toxicogenic  and  pathogenic  bacteria. 


CHAPTER  VI. 

The      Microscope. — The      Hanging      Drop.— Simple 

Staining 123 

Simple  and  compound  microscopes. Spherical  and  chro- 
matic aberration. Achromatic  and  apochromatic  objectives. 

— Under-correction. Magnifying-  power,  equivalent    focal 

•distance. Denning     power. Resolving    and    penetrating 

power. Numerical  aperture. Designation  of  objectives. 

Huyghenian    eye-piece. Compensating    eye-pieces. 

Designation  of  oculars. 

Abbe  condenser,  iris  diaphragm. Structural  and  colored 

images. Rules  for  use  of  condenser,  to  secure  illumination. 

The   stand,  graduation  of  draw- tube. Coarse   and  fine 

adjustment. Nose-piece. Care  of  microscope. 

Measurement  of  an  object. Stage  and  ocular  microme- 
ters.  Micromillimeter  or    micron. Micrometer   value    of 

an  objective. 

Slides  and  cover-glasses. Cleaning  of    the    latter. 

Cover-glass  forceps  for  bacteriological  work. 

Examination  of  living  bacteria. The  hanging-drop. 

Laboratory  work. 

Staining  of  bacteria. Acid  and   basic  anilin  dyes. — 

Stock  solutions. Dilute  stains. Simple  staining. Labor- 
atory work. 

CHAPTER   VII. 

Gelatin  and  Potato  Media, — Cultivation  of  Bacteria    .      152 

Preparation  of  the  meat  extract. Alkalization. 

Cleaning  and  plugging  of  tubes. Dry  and  moist  heat  steriliza- 
tion.  Fractional  sterilization  at  100°,  at  60°. Steam  steril- 
izer.  The  autoclave. 

Preparation  of  potato  media.-- — Dilution  and  mass  cul- 
tures.  The  pure  culture. Precautions  in  work. Labora- 
tory work. 

Gelatin  plate  cultivation. Advantages  over  potato  and 

liquid  media. Glass  plates. -Platinum  wires. Dilution  in 

gelatin. Pouring  on  plates. Plating  apparatus. Moist 

chambers. Apparatus  for  constant  low  temperature. 

Laboratory  work. 

Modified  gelatin  plate  method. Petri  dishes.- — Esmarch 

roll-tubes. Laboratory  work. 


10  CONTENTS. 

Modified  potato  cultures. Esmarch  dishes. Potato 

tubes. Preparation  of  Roux  tubes. Laboratory  work. 

Examination  of  colonies,  macroscopic  and  microscopic. 

Transplantation  of  colonies. Stab  cultures. The  plan  of 

study. Laboratory  work. 


CHAPTER    VIII. 

The  Non-Pathogenic  Bacteria   .          .          .          .          .193 

Bacillus  prodigiosus. B.   Indicus. B.   ruber  of  Kiel. 

B.   rubidus. B.  violaceus. B.  fluorescens  putidus. 

B.  phosphorescens. Orange  sarcine. Yellow  sarcine. B. 

subtilis. B.  mesentericus  vulgatus. B.  megaterium. B. 

ramosus. Proteus    vulgaris. Bacterium    Zopfii. Spiril- 
lum rubrum. B.  acidi  lactici. B.  butyricus. B.  cyano- 

genus. 

CHAPTER    IX. 

Bouillon,    Agar,    Milk    and    Modified     Media. — The 

Incubator  and  Accessories 232: 

Advantages  of  bouillon,  agar  and  milk. Preparation  of 

bouillon. Preparation  of  agar. Sterilization  of  milk. — 

Modified  media:  peptonless  agar,  glycerin  agar;  glucose,  lactose, 

litmus  and  serum  media. Laboratory  work. 

The     incubator. Thermo-regulators. Gas-pressure 

regulator. Micro-burners. Koch's  safety  lamp. Ther- 
mometers. 

CHAPTER   X. 

Relation  of  Bacteria  to  Disease. — Methods  of  Infec- 
tion and  Examination    ......     253- 

Infectious    diseases. Infection     and     intoxication. 

Rules    of    Koch. Generation. Attenuation. Bacterial, 

fungous  and  protozoal  diseases. Unknown  causes. 

Methods  of  infection. Cutaneous  application. Subcu- 
taneous injection. Animal  holders. Intravenous  injection. 

Intraperitoneal. Intrapleural. Anterior    chamber    of 

the     eye. Lymphatics. Intracranial. Infection     along 

respiratory  tract. Alimentary  tract. 

Observation  of  infected  animals. Keeping  of  record.— 

Food  and  drink. 

Post-mortem  examination. Sterilizing  case  for  instru- 
ments.  Preparation  of  the  animal. Examination  of  the 

eub-cutis. Peritoneal    and    pleural  cavities. Removal  of 


CONTENTS.  11 

portions  of  organs. Drawing"  of  heart  blood. Cover-glass 

preparations  of  tissues  and  organs,  of  blood. Staining  of  the 

streak  preparations. Precautionary  measures. 

Laboratory  work  with  anthrax  animal. Gelatin  plates. 

Preparation  of  agar  plates. Impression  preparation  of  col- 
onies.  Hanging-drop  examination  of  blood. Simple  -stains 

of    streak    preparations. Gram's     method. Anilin-water 

gentian    violet. Spore      staining. Carbolic     fuchsin. 

Phagocytes. Summary. 


CHAPTER    XI. 

The  Pathogenic  Bacteria 295 

Bacillus   anthracis. B.    anthracis    symptomatic! B. 

oedematis  maligni. B.  oedematis  maligni  No.  II. B.  tetani. 

Culture  of  anaerobic  bacteria. Staining  of.flagella. 

Giant  whips.— B.  lepras. B.  tuberculosis. B.  mallei. 

B.  diphtheriae. M.  pneumonias  crouposas. B.  pneumonias. 

B.  rhinoscleromatis. Vibrio  choleras  Asiaticas. Vibrio 

Deneke. Vibrio   Finkler-Prior. Vibrio    Metschnikovi. 

B.  coli  communis. B.  typhosus, B.  icteroides. B.  pestis 

bubonicas. B.  influenzas. B.  pyocyaneus. Streptococcus 

pyogenes.— — Staphylococcus  pyogenes  aureus. Micrococcus 

gonorrheas. M.    tetragenus. Spirillum  Obermeieri. B. 

choleras  gallinarum. B.  choleras  suis. B.  rhusiopathias  suis. 

B.  muriepticus. 


CHAPTER    XII. 

Yeasts,  Moulds  and  Streptotrices         ....     385 

Yeasts,  size  and  multiplication. Saccharomyces. 

Torula  or  wild  yeasts.— — Pathogenic  forms. 

Moulds.  Mycelium. Hyphas. Fruit-organs. 

Characteristics  of  the  mucor,  aspergillus,  penicillium,  oidium, 

Relation  to  fermentation. Pathogenic  forms. Skin 

diseases. 

Streptotrices. Relation  to  moulds  and  to  bacteria. 

Laboratory  work. Preparation  of  bread  flasks. Exam- 
ination of  moulds. 

Saccharomyces  cerevisias. S.  glutinis. Oidium  lactis. 

Monilia  Candida. Mucor  corymbifer. M.  rhizopodi- 

formis. Aspergillus  niger. A.  flavescens. A.  fumigatus. 

— Penicillium  glaucum. Achorion  Schonleinii. Strep- 

tothrix  actinomyces. S.  Maduras. S.  farcinica. 


12  CONTENTS. 

CHAPTER    XIII. 

Examination  of  Water,  Soil  and  Air   ....      422 

Water  as  a  carrier  of  disease. Limitations  of  a  chemi- 
cal   analysis. Recognition    of     typhoid    bacilli. Cholera 

vibrios. Method  of  analysis. Counting-  of  colonies,  direct 

and  by  aid  of  microscope. Number  and  kind  of  species. — 

Detection  of  pathogenic   bacteria  by  animal  inoculation. — 
Bacterial  contents  of  snow,  ice,  rain-water,  lakes,  rivers,  wells 
and  sea-water. 

The  bacteria  of  the  soil. Their  action. Pathogenic 

forms. Effect  of  rain  and  snow. Filtering  action  of  'the 

soil. Method  of  analysis. 

The  source  of  air  bacteria. Effect  of  moist  surfaces. — 

Purification  of  air  by  sedimentation. By  rain  and  snow. — 

Expired  air. Number  of  organisms  in  the  air.— —Country  and 

city  air. Pathogenic  bacteria. Methods  of  analysis. — 

Laboratory  work. 

CHAPTER  XIV. 

i 

Special  Methods  of  Work    ....          .          .     456 

Making  of    Pasteur  pipettes. Drawing  of   blood   from 

animals,  from  man. Oxalate  blood  and  plasma. Prepara- 
tion of  blood-serum. Sterilization  of  serum  by  filtration. — 

Fractional  sterilization  at  58°,  at  75°,  at  100°. Solidification 

of  serum. Modified  serum  media. Filtration  of  bacterial 

liquids. Tuberculin. Diphtheria    toxin. The    minimum 

fatal  dose. Testing  of  antitoxin. Immunization    against 

diphtheria. Antitoxic  and   anti-infectious  serum. Active 

and     passive      immunity. Pfeiffer's     reaction. Eisner's 

medium. Stoddart's  medium. Hiss'  tube  medium. Usch- 

insky's  medium.- — Preparation  and  use  of  collodium  sacs. — 
Inoculation  for  rabies. 

CHAPTER    XV. 

Special  Methods  of  Work,  Continued     ....     505 

Serum   agglutination. Poisonous    foods. Purification 

of  litmus. The  tubing  of  media.— : — The  sealing  and  keeping 

of  cultures. Thermal  death  point. -Moist  and  dry  heat. 

Testing  of  disinfectants. Testing   of   antiseptics. Room 

disinfection. Hardening,  imbedding  and  cutting  of  sections. 

The  staining  of  sections. Simple,  and  Gram's  method. 

Staining  of   anthrax,  tubercle  leprosy  and  typhoid  bacilli,  and 
actinomyces  in  sections. 

List  of  apparatus  and  chemicals    .         .         .         .         .  ;      .      547 
Index  ...  549 


LIST  OF  ILLUSTRATIONS. 


Frontispiece  showing-  component  parts  of  a  microscope.  PAGE. 

Fig.  1. — Micrococcus,  bacillus,  spirillum 17 

2. — Bacterium,  vibrio,  spirochaete 18 

3. — Involution  forms ' 21 

4. — Plasmolytic  changes 27 

5.— Capsulated  bacteria 29 

6. — Motile  organs  or  whips  on  bacteria 36 

7.— Giant-whips 39 

8.— Cell  division 42 

9. — Bacilli,  single,  in  pairs  and  in  threads 43 

10. — Division  forms  of  micrococci 44 

11.— Spirillum  forms : 46 

12.— Sporulation 49 

13. — Position  of  spores  and  resultant  forms 50 

14.— Spore  germination 54 

15.— Virtual  image,  simple  microscope  (Carpenter) 123 

16.— Real  image  (Carpenter) 124 

17. — Principle  of  the  compound  microscope  (Carpenter) 125 

18.  —Arrangement  of  lenses  in  an  objective.    Angle  of  aper- 
ture (Carpenter) 130 

19. — Structure  and  action  of  the  Abbe  condenser 133 

20.— Cover-glass  forceps  (F.  G.  N.) 141 

21.— The  "  hanging-drop  " 143 

22.— Water-bath  with  hot  iron  plate  (F.  G.  N.) . .  150 

23.— Enamelled  jar  for  making  media '. 153 

24.— Burettes  for  titrating  media  (F.  G.  N.) 155 

25.— Dry-heat  sterilizer 160 

26.— Steam  sterilizer  (F.  G.  N.) 164 

27.— Autoclave 165 

28, — Apparatus  used  in  cultivation 172 

29.— Inoculation  of  a  single  tube. 173 

30.— Inoculation  from  tube  to  tube  when  diluting 174 

31. — Ice  apparatus  for  cooling  plates 176 

32.— Water  apparatus  for  cooling  plates  (F.  G.  N.) 177 

33.— Constant  low  temperature  apparatus  (F.  G.  N.) 179 

34. — Potato  tubes,  ordinary  and  Roux  form 184 

35.  —Apparatus  for  filtering  agar  (F.  G.  N. ) 237 

36.— The  incubator  or  thermostat. .  244 


14  LIST  OF  ILLUSTRATIONS. 

37.— Thermo-reg-ulator  (F.  G.  N.) 246 

38.— Murrill's  gas  pressure  regulator 250 

39.— Koch's  safety  burner /  251 

40.— Adjustable  syringe. 262 

41. — Syringe  and  holder,  water-bath  and  radial  burner 263 

42. — Injection  apparatus 264 

43.— Sterile  conical  test-glass 265 

44. — Voges'  cylindrical  holder .' 266 

45.— Latapie's  animal  holder. 268 

46.— Rat  cage  and  forceps 273 

47. — Vaughan's  cage  for  rabbits,  etc 274 

48.— Instrument  sterilizing  case  and  searing  iron 275 

49.— The  Roux  spatula  and  Nuttall  needle 278 

50.— Simple  bottle  for  anaerobes  (F.  G.  N.) 308 

51.— Bottle  for  tube  culture  of  anaerobes  (F.  G.  N.) 314 

52. — Apparatus  for  plate  cultivation  of  anaerobes  (F.  G.  N.)  314 

53. — Apparatus  for  plate  cultivation  of  anaerobes  (F.  G.  N.)  314 

54.— Tube  culture  of  tubercle  bacillus  on  potato  (F.  G.  N.). .  315 

55.— Yeast    cells    with   spores   (Hansen) 387 

56. — Fruit-organs  of  moulds  (Lehmann) 390 

57.—  Wolff hugel's    counter 434 

58.— Laf  ar's  and  Jeffer's  counters  436 

59. — Esmarch's  roll- tube  counter 448 

60. — Apparatus  for  the  examination  of  air 454 

61. — Drawn-out  tube  pipettes  of  Pasteur 457 

62. — Sealing  cultures  in  capillaries 459 

63. — Pipette  used  in  drawing  blood 460 

64.— Roux  water-bath  for  serum  sterilization ." 466 

65. — Koch's  serum  sterilizer 467 

66.— Apparatus  for  filtering  bacterial  liquids  (F.  G.  N.) 469 

67. — Connections  for  filtering  apparatus  (F.  G.  N.) 470 

68— Berkefeld  filter;  globe  receiving  flask  (F.  G.  N.) 471 

69. — Roux  flask  for  surface  cultures 486 

70.— Rolling  of  collodium  sacs 497 

71.— Collodium  sacs 499 

72.— Tubing  of  media  (F.  G.  N.) 511 

73.— Keeping  of  cultures  (F.  G.  N.) 512 

74. — Filter  for  bacterial  suspensions 514 

75.— Determination  of  the  thermal  death-point  (F.  G.  N.).  . .  516 

76. — Formaldehyde  apparatus  for  room  disinfection  (F.  G.N)  530 


CHAPTER   I. 

FORM  AND  CLASSIFICATION  OF  BACTERIA. 

/ 

Bacteria  may  be  characterized  as  unicellular,  micro- 
scopic plants.  Their  vegetable  nature  has  been  established 
only  within  comparatively  recenl;  f^a^i'  ,Frc*P  £ne  time  of 
Leeuwenhoek,  who  in  1683  discovered  .the  first,  representa- 
tive of  this  group,  until  1854  „  these  microscopic'  organisms 
were  spoken  of  as  animalculae  or  minute  forms  of  animal 
life.  Indeed,  it  was  not  until  about  1875  that  the  true 
relationship  of  bacteria  to  plants  was  definitely  settled, 
largely  through  the  labors  of  Cohn,  Naegeli,  DeBary  and 
other  botanists. 

When  studying  the  exceedingly  minute  forms  of  life 
observed  under  the  microscope,  one  recognizes  that  it  is  not 
always  possible  to  indicate  the  dividing  line  between  the 
animal  and  vegetable  kingdoms.  There  is  no  one  charac- 
teristic which  will  serve  for  this  purpose.  Prom  the  very 
nature  of  things  it  must  be  expected  that  the  lowest  forms 
of  animal  and  plant  life  will  approach  one  another.  The 
striking  differences  which  are  seen  between  the  higher 
forms  of  animal  and  plant  -life  gradually  disappear  as  the 
comparisons  are  carried  down  the  scale  to  the  lowest  forms. 
The  latter  merge  into  one  another  indicating  a  common 
origin  in  the  remotest  past.  Under  these  conditions  it  is 
evidently  impossible  to  characterize  certain  forms  of  life  as 
plants  or  as  animals. 

Inasmuch  as  bacteria  belong  to  the  lowest  and  simplest 
forms  of  life,  it  cannot  be  expected  that  they  will  show  any 
marked  differentiation  into  plants.  They  are  classified, 
however,  among  plants  because  of  their  evident  relation. 


16  BACTERIOLOGY. 

ship  to  certain  well  recognized  types  of  plant  life.  In  their 
characteristics  of  growth,  multiplication  and  reproduction 
they  resemble  the  group  of  algse  more  than  any  other  group 
of  living  beings,  and  it  is  this  general  relationship,  rather 
than  any  one  peculiarity,  which  has  led  to  their  being 
placed  in  the  vegetable  kingdom. 

As  will  be  indicated  later,  bacteria  multiply  by  division 
or  fission,  and  for  this  reason  they  are  spoken  of  as  fission- 
fungi  or  schizomyc$te$.f  4  The  word  germ  or  microbe  is  often 
employed  to  'designate;  bacteria.  It  should  be  understood, 
however^  thai ;  titese  jtsva,  tftrriis  have  a  broader  significance 
and  inciude  all  microscopic  life,  whether  animal  or  plant. 
Bacteria  therefore  constitute  a  definite  group  of  germs  or 
microbes. 

In  addition  to  their  relationship  to  algae,  the  bacteri  i 
resemble  in  many  respects  the  moulds  or  fungi,  and  the 
yeasts.  These  two  groups  will  be  discussed  more  in  detail 
in  a  subsequent  chapter.  It  will  be  sufficient  to  indicate  in 
this  connection  the  more  marked  distinctions  between  the 
moulds,  yeasts  and  bacteria.  The  former,  as  is  well 
known,  appear  in  velvety  or  cotton-like  spreading  growths 
which  even  the  unaided  eye  can  often  resolve  into  a  net- 
work of  threads.  The  moulds  are  therefore  spoken  of  as 
thread  fungi  or  hyphomycetes.  Growth  results  not  by 
division,  as  in  the  case  of  bacteria,  but  by  the  lengthening 
of  a  cell  or  thread,  that  is  to  say  by  continuous  end-growth. 
Moreover,  the  moulds  are  relatively  higher  forms  of  plant- 
life  inasmuch  as  many  species  of  this  group  possess  special 
fruit-organs.  The  threads  which  make  up  the  mass  of  a 
mould  are  much  thicker  than  bacterial  cells. 

The  yeasts  are  uni-cellular  plants  which  differ  from 
bacteria  in  size,  method  of  multiplication  and  in  other 
respects.  The  yeast  cell  may  be  considered  as  a  giant  in 
comparison  with  the  minute  bacterial  cell.  Moreover, 
yeasts  multiply,  not  by  fission,  but  by  a  process  known 


FORM  AND  CLASSIFICATION  OF  BACTERIA.  17 

as   budding.      They  are  designated  by  the  term  blastomy- 
cetes. 

A  great  many  elaborate  attempts  have  been  made  at 
classifying-  bacteria.  Owing  to  their  extremely  minute  size 
it  is,  as  a  rule,  impossible  to  follow  out  the  life-history  of 
each  individual  species.  The  characteristic  development 
of  the  fruit-organs  in  higher  plants  affords  a  basis  for  a 
natural  classification  whereby  the  various  species  are 
grouped  into  genera  and  families.  Such  a  classification  is 
natural,  because  it  brings  together  the  various  individuals 
which  possess  the  same  structure  and  development.  Inas- 
much as  bacteria  are  unicellular  it  follows  that  they  do  not 
possess  definite  fruit-organs,  and  owing  to  their  size  very 
little  indeed  can  be  said  of  their  structure.  The  various 
classifications  proposed  are  based  upon  characteristics  such 
as  form,  size,  manner  of  division,  presence  of  spores, 
motion,  number  and  arrangement  of  whips,  etc.  It  is  evi- 
dent, therefore,  that  all  such  systems  of  classification  are 
more  or  less  artificial. 

For  practical  purposes  it  is  sufficient  to  divide  bacteria 
according  to  their  external  form  into  three  groups.  These 
are: 

Micrococci,  or  spherical  bacteria; 

Bacilli,  or  rod-like  bacteria; 

Spirilla,  or  screw-shaped  bacteria. 


c 

FIG.  i.    a— Micrococcus;    £— Bacillus;    <:— Spirillum. 

In  a  few  instances  special  names  are  applied  to  certain 
forms  of  one  or  another  of  these  three  primary  types. 
Thus,  the  term  bacterium  is  occasionally  applied  to  a  very 
short  bacillus.  It  has  the  same  significance  as  the  word 

cocco-bacillus,  which  indicates  that  the  organism  may  at 
2 


18  BACTERIOLOGY. 

times  be  almost  spherical,  at  other  times  rod-shaped. 
Again,  the  term  vibrio  is  applied  to  certain  bacteria  which 
may  form  spirals,  but  which  commonly  grow  in  segments 
of  a  spiral.  They  may  therefore  be  considered  as  bent, 
twisted  rods  which  appear  under  the  microscope  as  comma- 
like  forms.  The  union  of  two  of  these  "comma  bacilli" 
gives  an  elongated  S  form,  and  when  more  of  these  elements 
unite  thus  they  give  rise  to  a  spirillum.  Long,  slender, 
flexible  spirals  are  frequently  designated  as  spirochcetes. 
Additional  terms  will  be  met  with  when  describing  the 
various  characteristics  of  growth  of  bacteria. 


FIG.  2    a — Bacterium;    b — Vibrio;    c — Spirochaete. 

The  above  classification  is  based  upon  the  external 
forms  of  bacteria,  and  is  therefore  artificial  in  character. 
A  similar  classification  applied  to  the  higher  forms  of 
life  would  lead  to  gross  error.  Thus,  the  worm,  eel  and 
snake,  viewed  at  a  distance,  show  the  same  general  appear- 
ance, and  yet  they  are  wholly  different,  unrelated  types  of 
life.  A  close  inspection  at  once  reveals  striking  differences. 
In  the  case  of  bacteria,  owing  to  the  extremely  small  size 
and  the  simplicity  of  the  bacterial  cell,  it  is  not  possible  to 
establish  structural  differences,  and  for  that  reason  the 
form  classification,  imperfect  as  it  may  be,  is  of  necessity 
adopted. 

The  various  species  of  micrococci  will  show  differences 
in  their  size,  that  is  in  their  diameter,  but  otherwise  they 
closely  resemble  one  another.  Occasionally,  when  micro- 
cocci  grow  in  pairs  they  may  show  flattened,  apposed  sur- 
faces, as  if  two  biscuits  were  brought  together.  In  other  in- 
stances they  may  be  elongated  or  lance-shaped  (see  fig.  10  a). 
In  the  rod-shaped  bacteria  or  bacilli  greater  differences  will 


FORM  AND  CLASSIFICATION  OF  BACTERIA.  19 

be  observed  in  the  form  and  size  of  various  species.  Thus, 
one  species  may  be  very  short  and  thick,  while  another 
may  be  considerably  longer  and  narrower,  or  longer  and 
thicker.  Again,  in  one  species  of  bacillus  the  ends  may  be 
square,  while  in  others  they  may  be  rounded  or  ellipsoidal. 
Among  the  spirilla  marked  variations  in  form  or  size  will 
be  observed.  So  much  so  that  it  is  very  doubtful  at  times- 
whether  certain  species  really  belong  to  the  group  of  bac- 
teria. 

While  some  species  of  bacteria  show  marked  differences 
in  form  and  size,  it  must  not  be  supposed  that  this  is  always 
the  case.  On  the  contrary  many  undoubtedly  distinct 
species  of  bacteria  may  have  the  same  form  and  size,  so 
that,  as  far  as  the  microscope  is  concerned,  they  cannot  be 
distinguished  one  from  another.  The  division  of  micro- 
cocci,  bacilli  and  spirilla  into  species  is  based,  as  a  rule, 
not  upon  the  mere  microscopical  appearance,  but  rather 
upon  the  sum  total  of  the  properties  they  possess.  The 
characteristics  of  growth  on  various  artificial  media,  the 
behavior  to  staining  reagents,  and  the  effect  on  the  living 
animal  must  all  be  considered  when  an  attempt  is  made  to 
to  distinguish  one  species  from  another.  For  this  reason  it 
is  not  possible,  except  in  very  few  instances,  to  identify 
species  among  bacteria  by  mere  microscopic  examination. 
In  the  majority  of  cases  the  identification  of  species  neces- 
sitates a  careful,  long  and  tedious  study  of  all  the  proper- 
ties possessed  by  that  organism. 

The  higher  forms  of  plant  and  animal  life  possess,  as  a 
rule,  a  constant  form  and  size.  Marked  changes  in  the 
environment,  such  as  temperature,  and  altitude,  are  neces- 
sary to  produce  type  variations.  Bacteria,  however,  are 
extremely  liable  to  undergo  alterations  in  form,  size  and 
other  characteristics.  Owing  to  their  simple  nature  they 
are  readily  influenced,  in  some  way  or  another,  by  the 
slightest  change  in  their  environment.  A  variation  of  a 
few  degrees  in  temperature,  a  trifling  difference  in  the 


20  BACTERIOLOGY. 

reaction  or  chemical  composition  of  the  soil,  may  profoundly 
affect  the  form  and  size  of  bacteria. 

It  is  evident,  therefore,  that  a  given  species  of  bacteria 
does  not  possess  a  constant  form  or  size.  Under  certain 
conditions  of  environment  it  may  be  a  large,  thick  bacillus, 
while  at  other  times  it  may  appear  almost  like  a  coccus. 
By  the  typical  form  of  a  given  species  is  meant  that  form 
which  is  met  with  when  the  best  conditions  of  temperature, 
soil,  oxygen  supply,  etc.,  are  provided.  The  slightest 
variation  from  these  optimum  conditions  will,  as  indicated 
above,  cause  a  deviation  from  the  typical  form. 

Variations  in  form  and  size  may  be  considered  as 
arising  from  natural  causes,  the  environment;  and  from 
artificial  causes  due  to  methods  of  manipulation. 

When  a  perfectly  pure  specimen  is  examined  under  the 
microscope  more  or  less  marked  differences  in  the  size  of 
the  various  cells  will  be  observed.  Such  differences  must 
be  expected  among  living,  actively  growing  forms.  The 
small,  young  cells  will  always  be  present  beside  the  large, 
old  cells.  Again,  most  of  the  bacterial  cells  in  a  given 
specimen  may  be  single,  but,  now  and  then,  some  will  be 
found  forming  threads,  or  filaments,  many  times  longer 
than  the  single  cell.  These  thread-like  forms  are  not  con- 
taminations due  to  the  presence  of  a  different  species,  as 
might  at  first  thought  be  supposed. 

Another  variation  may  be  expected  when  the  adult 
cell  develops  a  spore  or  seed.  Very  characteristic  forms 
result  from  the  presence  of  a  spore  within  the  bacterial 
cell.  These  will  be  given  special  attention  in  a  subsequent 
chapter. 

The  composition  of  the  medium  on  which  the  bacteria 
grow  will  exert  a  marked  influence  upon  their  form  charac- 
teristics. The  s'ame  organism  planted  on  solid  media,  such 
as  coagulated  blood  serum,  agar  and  potato,  will  show  dif- 
ferences in  form.  The  addition  of  small  amounts  of  glycerin- 


FORM  AND  CLASSIFICATION  OF  BACTERIA.  21 

glucose,  or  an  acid,  or  alkaline  reaction  likewise  may  affect 
the  appearance  of  the  cell. 

A  comparison  of  the  growths  on  solid  and  on  liquid 
media  will  show  peculiarities  and  modifications  in  the  form 
and  size  of  a  given  species.  Thus,  single  cells'  or  at  most 
short  threads  may  predominate  on  solid  media,  whereas  in 
liquid  media  very  long  threads  or  filamentous  growths  may 
be  found. 

The  temperature  exerts  a  profound  effect  upon  the 
form  characteristics  of  bacteria.  At  low  temperature  the 
growth  is  slower,  and  hence  the  individual  cell  may  attain 
an  unusual  size,  whereas  at  a  higher  temperature  multipli- 
cation results  so  rapidly  that  the  cells  are  considerably 
smaller. 

Under  unfavorable  conditions  of  soil  or  temperature 
certain  bacteria  will  show  remarkable  variations  from  the 
normal  type.  What  is  ordinarily  a  perfect  rod  becomes 
distorted  out  of  all  semblance  to  the  original  form.  Club- 
shaped,  spindle-shaped,  dumb-bell-like  forms  are  produced. 
Sometimes  they  become  twisted  into  irregular,  spherical 
bodies.  These  peculiar,  deformed  cells  are  considered  as 
degenerations.  They  are  commonly  designated  as  involu- 
tion forms.  The  organism  is  struggling  for  existence  under 
adverse  conditions  which,  if  they  persist,  will  eventually 
cause  its  destruction.  Transplantation  to  a  favorable 
medium  will  promptly  restore  the  typical  form. 


FIG.  3.    Involution  forms. 

The  alterations  mentioned  above  are  the  result  of 
environment.  Whatever  may  be  the  variation,  it  is  to  be 
considered  always  as  temporary,  inasmuch  as  the  typical 


22  BACTERIOLOGY. 

form  can  be  reproduced  whenever  the  organism  is  trans- 
planted and  grows  under  its  most  favorable  conditions  of 
soil  and  temperature. 

The  methods  of  examination  may  apparently  influence 
the  form  of  a  given  species.  This  is  very  often  seen  in 
stained  preparations.  As  a  result  of  heavy  staining,  such 
as  is  resorted  to  in  order  to  demonstrate  flagella,  the 
organism  appears  much  larger  and  thicker  than  when 
stained  in  the  ordinary  way.  The  successive  deposition  of 
the  dye,  layer  on  layer,  causes  an  apparent  increase  in  the 
size  of  the  cell.  Again,  in  the  method  of  double- staining 
tubercle  and  leprosy  bacilli,  the  bacteria,  after  being  heavily 
stained,  are  partly  decolored  with  alcohol.  Depending 
upon  the  extent  of  the  decoloration,  they  may  appear  as 
relatively  thick  or  as  extremely  thin  rods. 

As  a  rule  the  bacteria  present  in  tissues,  when  stained, 
appear  smaller  and  narrower  than  in  the  fresh  material. 
This  variation  is  in  part  due  to  the  contraction  of  the  pro- 
toplasm or  contents  of  the  cell  by  the  alcohol  employed  to 
harden  the  tissue.  It  is  also  due  to  partial  decoloration 
which  is  unavoidable  when  the  bacteria  are  to  be  differen- 
tiated from  the  tissue  in  which  they  lie  imbedded. 

As  a  result  of  the  action  of  various  chemical  substances 
the  protoplasm  of  the  bacterial  cell  may  be  contracted  or 
drawn  up  into  irregular  masses  or  granules.  When  such 
forms  are  stained  they  may  appear  like  minute  micrococci, 
although  the  original  form  was  that  of  a  typical  rod.  This 
action  on  protoplasm  is  designated  as  plasmolysis.  Expos- 
ure of  bacteria  to  iodine  is  likely  to  result  in  plasmolytic 
changes.  Similar  alterations  are  met  with  as  the  result  of 
overheating  specimens  in  the  process  of  preparation  (Fig.  4). 

From  what  has  been  said  above  with  reference  to 
variation  in  the  form  of  a  given  species,  it  is  evident  that 
pleomorphism  is  very  common  among  bacteria.  Certain 
species  are  more  prone  to  undergo  modifications  of  this 


FORM  AND  CLASSIFICATION  OF  BACTERIA.  23 

kind  than  others.  Variations  in  the  form  and  size  of  the 
individuals  of  a  given  species  must  be  expected.  The  same 
form  will  be  maintained  only  when  all  the  conditions  of 
environment  are  constant. 

In  view  of  the  marked  alterations  in  the  form  of  bac- 
teria, it  has  been  supposed  that  there  is  a  corresponding- 
variability  of  species.  Under  certain  conditions  of  environ- 
ment a  given  species  may  cease  to  produce  the  pigment 
which  under  normal  conditions  it  would  elaborate.  Again, 
similar  unfavorable  conditions  may  cause  a  disease-pro- 
ducing germ  to  lose  its  property  of  growing  in  the  living 
body.  In  other  words,  not  only  the  mere  form  and  size, 
but  also  the  physiological  activities  of  the  cell  may  be 
altered.  This  weakening  of  the  functional  activity  of  a 
bacterial  cell  is  known  as  attenuation.  The  attenuated  or 
weakened  germ  constitutes  a  variety  of  the  original  type, 
but  it  does  not  constitute  a  new  species.  On  reversing  the 
conditions  of  environment,  that  is  rendering  these  more 
favorable,  the  attenuated  form  can  be  brought  back  to  the 
full  possession  of  its  original  properties. 

Undoubtedly,  species  have  originated  in  the  past  as  a 
result  of  growth  under  altered  conditions,  and  new  species 
may  even  now  be  in  process  of  formation.  But  under 
ordinary  conditions  of  observation  this  change  of  one 
species  into  another  does  not  and  cannot  take  place.  The 
hay  bacillus  cannot  be  transformed  into  the  anthrax  bacil- 
lus, nor  can  the  germ  of  pneumonia  be  converted  into  that 
of  consumption.  They  may  both  be  greatly  modified, 
giving  rise  to  varieties,  but  so  long  as  these  exist  they  still 
represent  the  original  species.  The  typical  form  and  the 
typical  species  can  always  be  reproduced  by  restoring  the 
most  favorable  conditions  of  growth. 


CHAPTER    II. 
SIZE  AND  STRUCTURE  OF  THE  BACTERIAL  CELL. 

Bacteria  have  been  described  as  uni-cellular  micro- 
organisms. The  fact  that  they  consist  of  a  single  cell 
indicates  at  once  their  microscopic  character.  The  micro- 
scope reveals  a  world  of  animate  beings  which  differ 
enormously  in  size.  Some  of  these  organisms  may  be  large 
while  others  are  exceedingly  small.  The  bacteria  are 
usually  spoken  of  as  the  smallest  of  living  beings,  the 
" infinitely  small."  Until  very  recently  no  form  of  life  was 
known  smaller  than  these.  The  discovery  of  the  microbe 
of  pleuro-pneumonia  has  revealed  the  existence  of  organ- 
isms considerably  smaller  than  the  bacteria.  This  new 
organism  is  so  small  that  the  very  highest  magnification  of 
the  microscope  still  leaves  its  form  uncertain. 

The  size  of  microscopic  objects  is  usually  expressed 
in  micro-millimeters.  A  micro-millimeter  or  micron  may 
be  defined  as  the  thousandth  part  of  a  millimeter.  Inas- 
much as  a  millimeter  corresponds  approximately  to  sV 
of  an  inch,  it  follows  that  one  micro-millimeter  repre- 
sents 2tTi7T7  of  an  inch.  It  is  customary  to  speak  of  a 
micro-millimeter  as  a  micron  -and  to  designate  it  by  the 
Greek  letter  /*. 

The  largest  micrococcus  known  has  a  diameter  of  2  p.  or 
wm  of  an  inch.  There  are  some  micrococci  that  have  a 
diameter  only  one-tenth  as  large  as  this.  The  common 
pus-producing  micrococci  have  a  diameter  of  about  0.8  p.  or 
irtarF  of  an  inch.  Inasmuch  as  the  diameter  of  a  red  blood 
cell  is  about  8  /*,  or  about  rsW  of  an  inch,  it  is  evident  that 
ten  of  these  cocci  placed  in  a  row  would  correspond  to  the 


SIZE  AND  STRUCTURE  OF  THE  BACTERIAL  CELL.  25 

diameter  of  a  blood  cell.  The  average  micrococcus  has  a 
diameter  of  about  1  /;-,  or  sdnnr  of  an  inch. 

The  width  of  the  average  bacillus  is  about  1  ;j..  The 
length  will  vary  usually  from  2  to  4  //.  The  thickest  bacilli, 
like  the  B.  crassus,  are  said  to  have  a  width  of  4  p..  Usually 
several  bacilli  must  be  placed  end  to  end  to  correspond  to 
the  diameter  of  the  red  blood  corpuscle. 

From  the  dimensions  given  it  is  evident  that  an  enor- 
mous number  of  bacteria  may  be  present  in  a  relatively 
small  volume.  One  milligram  of  the  pure  cells  of  the 
golden  pus-producing  micrococcus  will  contain  about 
2,000,000,000  individuals.  One  grain  of  this  material  would 
therefore  contain  128,000,000,000  cells. 

In  view  of  the  extremely  minute  size  of  bacteria  it  is 
evident  that  they  are  very  close  to  the  limit  of  micros- 
copic vision.  It  is  manifestly  impossible  to  make  out  much 
of  any  structure  in  such  small  organisms.  The  ordinary 
plant  or  animal  cell  can  be  readily  shown  to  consist  of 
a  cell-wall,  a  protoplasm  and  a  nucleus.  It  may  there- 
fore be  expected  that  the  bacterial  cell  will  likewise 
consist  of  a  cell-wall,  containing  the  protoplasm  and  the 
nucleus. 

It  has  been  supposed  in  the  past  that  the  bacterial  cell 
possessed  a  cell-wall  which  did  not  stain  readily  and  which 
was  composed  of  cellulose  or  woody  fibre.  There  can  be 
no  question  regarding  the  existence  of  an  outer  membrane 
or  envelope,  but  its  chemical  composition  is  undetermined. 
Certain  sarcines  and  a  few  bacilli  have  been  said  to  give 
cellulose  reactions,  but  these  observations  have  not  been 
confirmed  by  subsequent  investigators.  Some  of  the  reac- 
tions employed  in  the  detection  of  cellulose  cannot  be 
considered  as  characteristic.  Thus,  the  production  of  a 
reducing  substance  on  treatment  with  an  acid  may  be  due 
to  cellulose,  but  it  may  also  be  due  to  the  presence  of  a 
glyco-proteid. 


26  BACTERIOLOGY. 

There  is  reason  to  believe  that  the  cell-wall  proper 
consists  chiefly  of  protein  substances.  The  cell-wall  may 
be  considered  as  a  layer  of  hardened  or  condensed  proto- 
plasm. It  is  stained  by  anilin  dyes  and  is  not  digested  by 
proteolytic  ferments  such  as  pepsin  and  trypsin.  It  appar- 
ently responds  to  Millon's  reagent,  but  is  insoluble  in 
Schweitzer's  reagent,  which,  as  is  known,  dissolves  cellu- 
lose. It  is  quite  possible  that  certain  species,  especially 
when  growing  on  special  media,  acquire  some  cellulose-like 
substance.  In  other  species  dextrin-like  products  seem,  to 
be  elaborated.  Again,  granulose,  or  a  chitinous  substance 
may  be  present  at  times  in  the  cell-wall. 

The  existence  of  a  cell-wall  is  indicated  by  various; 
characteristics  exhibited  by  bacteria.  Thus,  the  constant 
form  of  a  spirillum  or  of  rods  can  only  be  accounted  for  by 
the  presence  of  an  outer  hard  envelope.  Occasionally  the 
contents  of  a  cell  may  die  out  and  disappear,  in  which  case 
the  empty  shell  remains  and  is  easily  distinguished  from.! 
the  normal  cells,  which  stain  perfectly. 

The  presence  of  a  cell-wall  is  especially  clearly  demon- 
strated in  certain  cases.  Thus,  the  contents  of  the  cell 
contain,  in  addition  to  the  protoplasm,  more  or  less  of  ai 
watery  cell  fluid.  As  a  result  of  surface  tension  this  may 
gather  in  minute  droplets  between  the  semi-solid  proto- 
plasm. Occasionally  the  protoplasm  will  show  marked 
adhesion  to  the  wall,  and  as  a  result  the  fluid  is  prevented 
from  forming  globules  and  causes  a  separation  of  the  con- 
tents of  the  cell  into  discs.  A  disc  of  protoplasm  will 
alternate  with  a  disc  of  the  cell  fluid.  On  staining  such  an 
organism  it  will  exhibit  transverse  bands. 

The  cell  fluid  holds  in  solution  various  mineral  salts, 
which  exert  a  certain  osmotic  or  internal  pressure  which  is- 
counteracted  by  the  protoplasm  and  especially  by  the  firm 
outer  cell-wall.  The  normal  internal  pressure  of  the  cell 
contents  depends  upon  the  composition  of  the  fluid  sur- 
rounding the  cell.  When  the  cell  is  placed  in  water  which 


SIZE  AND  STRUCTURE  OF  THE  BACTERIAL,  CELL.  27 

contains  relatively  less  osmotic  substances  than  the  cell 
itself,  there  is  a  tendency  for  the  cell  fluid  and  the  dissolved 
substances  to  pass  out  into  the  surrounding-  water.  This  is 
prevented  by  the  outer  layer  of  protoplasm,  and  hence  the 
osmotic  or  internal  pressure.  When,  however,  the  cell  is 
placed  in  a  liquid  which  is  richer  in  osmotic  substances,  a_ 
2.5  per  cent,  saltpeter  or  a  1  per  cent,  salt  solution,  the 
osmotic  pressure  of  the  outside  liquid  overcomes  that  of 
the  cell  fluid.  A  current  is  thus  established  into  the  cell. 
The  protoplasm  as  a  result  retracts  from  the  cell  wall,  and 
this  retraction  of  protoplasm  is  known  as  plasmolysis. 


FIG.  4.     Plasmolytic  changes,  after  A.  Fischer.      a—  Cholera 
vibrio;    £—  Typhoid  bacillus;    c—  Spirillum  undula. 


In  the  above  case  the  salts  in  the  outer  liquid,  owing-  to 
the  relatively  impermeable  wall,  do  not  easily  penetrate 
into  the  cell.  If  they  did  the  internal  pressure  of  the  cell 
fluid  would  soon  rise  above  that  of  the  surrounding  liquid, 
and  as  a  result  the  protoplasm  would  expand  and  refill  the 
cell.  This  actually  does  occur  when  a  saltpeter  solution  of 
double  the  strength  given  is  employed.  When  the  plasmo- 
lyzed  cell  is  placed  in  pure  water  the  protoplasm  returns 
promptly  to  its  original  position. 

In  the  case  of  the  micrococcus  the  protoplasm  contracts 
to  a  small  globular  mass,  while  in  the  bacillus  two  round 
bodies  form,  one  at  each  end.  As  a  result  the  cell-wall 
between  the  polar  bodies  is  rendered  visible.  These  plas- 
molytic  'changes  occur  in  the  living  cell.  As  will  be  seen 
later,  the  flagella  or  motile  organs  seem  to  arise  from  the 
outer  layer  of  the  cell-wall,  in  which  case  this  membrane  is 
functionally  active  and  is  not  a  passive  structure,  as  in  the 
case  of  the  cellulose  wall  of  the  ordinary  plant  cell. 


28  BACTERIOLOGY. 

The  presence  of  a  cell-wall  is  also  demonstrated  by  the 
action  of  iodine  on  the  t  cells.  An  organism  treated  with 
iodine  solution  and  then  fixed  and  stained  will  show  the  cell 
membrane  more  or  less  distinctly.  The  protoplasm  has 
contracted  from  the  wall  and  a  number  of  colorless  globules 
or  vacuoles  will  be  seen,  due  to  the  cell  fluid  that  has  been 
squeezed  out.  Moreover  one  or  more  heavily  stained 
roundish  bodies,  or  "chromatin  granules,"  can  also  be 
observed. 

There  is  abundant  evidence,  therefore,  which  shows 
that  a  definite  membrane  is  present  in  all  the  stages  of 
the  development  of  the  bacterial  cell.  Moreover,  plasmo- 
lytic  experiments  indicate  that  the  protoplasm  is  not  firmly 
united  to  this  wall.  The  fact  that  strong  solutions  of  salts 
do  penetrate  the  interior  of  the  cell  indicates  that  the  cell- 
wall  is  permeable.  This  of  course  is  necessarily  so,  since 
the  nourishing  material  present  in  the  surrounding  fluid 
must  pass  through  this  wall  in  order  to  reach  the  proto- 
plasm. Similarly  the  waste  products  of  the  cell  must  pass 
outward  through  a  permeable  cell-wall. 

The  bacterial  membrane  or  cell-wall  is  usually  very 
thin  and  colorless,  and  for  this  reason  cannot  be  ordinarily 
seen.  As  indicated  above,  it  probably  consists  of  a  protein 
and  not  of  cellulose.  Under  special  conditions  the  outer 
layer  of  the  cell-wall  takes  up  water  and  gelatinizes.  In 
this  case  the  cell  becomes  surrounded  by  a  broad,  colorless 
zone  which  does  not  stain.  This  softened,  expanded  cell- 
wall  is  known  as  the  capsule.  Owing  to  its  soft,  slimy 
character  it  causes  the  cells  to  stick  together,  .thus  giving 
rise  to  masses  of  cells  which  are,  as  it  were,  cemented 
.together.  Such  a  mass,  when  touched  with  a  wire,  can  be 
drawn  out  into  long,  slimy  threads.  This  massed  condition 
of  certain  bacteria  is  designated  as  a  zooglea. 

Well  marked  capsule  formation  is  met  with  in  only 
comparatively  few  bacteria.  In  the  majority  of  these 
organisms  it  is  either  very  feebly  developed  or  entirely 


SIZE  AND  STRUCTURE  OF  THE  BACTERIAL  CELL. 


absent.  Capsulated  forms  are  met  with  most  often  when 
staining  the  bacteria  that  may  be  present  in  the  fluids  of 
the  animal  body.  Such  forms  are  therefore  met  with  oc- 
casionally in  saliva,  sputum  and  in  blood.  Certain  species, 
however,  may  give  rise  to  pronounced  capsules,  when  grown 
on  artificial  media  (Fig.  5  a). 


FIG.  5.  Capsulated  bacteria,  drawn  with  camera  lucida,  Zeiss  one- 
twelfth  and  ocular  2.  a — Anaerobic  bacillus  isolated  from  human  feces, 
capsules  formed  on  glucose  agar;  b — Micrococcus  tetragenus  with  cap- 
sules, from  blood  of  mouse — compared  with  red  blood  cell;  c — Lanceolate 
diplococcus  of  pneumonia  with  capsules — above  this  a  red  blood  cell  show- 
ing false  capsule,  due  to  shrinkage  in  drying. 

The  chemical  composition  of  the  soil  influences  the 
formation  of  the  capsule.  Thus,  the  leuconostoc,  when 
grown  on  sugar  media,  develops  remarkably  large  capsules, 
whereas  in  the  absence  of  sugar  this  gelatinization  of  the 
cell- wall  does  not  take  place.  It  is  evident  that  the  capsule 
is  not  a  degenerative  product,  but  a  normal  reaction 
induced  by  certain  constituents  of  the  medium. 

While  the  presence  of  capsules  gives  rise  to  slimy 
growths,  it  must  not  be  inferred  that  they  are  the  invariable 
cause  of  a  slimy  consistence.  Bacteria  may  actually  secrete 
a  mucin-like  sticky  substance,  and  as  a  result  the  liquid 
will  be  slimy  although  no  capsules  will  be  found  surround- 
ing the  individual  cells.  Instances  of  slimy  milk,  beer, 
wine,  etc.,  from  causes  of  this  kind  are  not  uncommon. 

The  detection  of  the  presence  of  a  capsule  is  not  always 
an  easy  matter.  Frequently,  as  a  result  of  manipulation 
capsule-like  forms  may  be  met  with.  This  is  invariably 
the  case  when  the  bacteria  are  present  in  an  albuminous 


30  BACTERIOLOGY. 

fluid  like  blood.  The  organic  matter  dries  out  on  the  cover- 
glass  first,  whereas  the  bacterial  cell  dries  later.  In  the 
process  of  drying  it  naturally  shrinks  somewhat  in  size, 
and  hence  a  clear  zone  results  between  the  dried  organic 
matter  and  the  retracted  dried  cell.  On  staining  the  speci- 
men a  colorless  zone  will  surround  each  cell  and  may  there- 
fore be  easily  mistaken  for  a  capsule  (Fig.  5  c).  The 
capsule,  unlike  the  real  membrane,  does  not  stain  easily 
with  anilin  dyes. 

In  certain  organisms  related  to  the  bacteria,  as  the 
beggiatoa,  a  hardening  rather  than  softening  of  the  mem- 
brane takes  place.  This  gives  rise  to  a  sheath  or  tube 
which  surrounds  the  individual  elements  and  may  be  consid- 
ered as  analogous  to  the  cell-wall  of  higher  plants.  Such 
sheaths,  however,  are  not  met  with  among  the  true  bacteria. 

The  contents  of  the  bacterial  cell.  The  relatively  hard  and 
more  or  less  impenetrable  cell-wall  encloses  the  soft  proto- 
plasm which  is  the  living  portion  of  the  cell.  In  it,  there- 
fore, are  carried  on  the  chemical  and  physical  changes 
necessary  to  life.  It  contains,  like  all  protoplasm,  a  rela- 
tively large  amount  of  water.  The  solid  constituents  of  the 
protoplasm  are  chiefly  nitrogenous,  that  is  to  say  protein, 
in  character.  Moreover,  fatty  substances  are  always  pres- 
ent, and  in  some  species,  as  the  tubercle  bacillus,  they  may 
make  up  a  large  percentage  (30-40  per  cent.)  of  the  total 
solids.  There  is  reason  to  believe  that  at  times  a  carbohy- 
drate, like  granulose,  is  present.  The  inorganic  constitu- 
ents, or  ash,  constitute,  about  10  per  cent,  of  the  dried 
cells. 

The  composition  of  the  bacterial  cell  will  vary  accord- 
ing to  the  soil  on  which  it  grows.  Moreover,  it  is  undoubt- 
edly influenced  by  the  age  of  the  individual  cell.  In  view 
of  the  different  products  elaborated  by  various  species  of 
bacteria,  it  is  evident  that  their  chemical  composition  must 
necessarily  vary. 


SIZE  AND  STRUCTURE  OP  THE  BACTERIAL  CELL.  31 

The  higher  animal  and  plant  cell  always  contains  a 
nucleus  within  the  protoplasm.  The  nucleus  unquestion- 
ably plays  a  most  important  part  in  the  division  of  the  cell. 
.So  much  so  that  it  has  been  considered  essential  to  repro- 
ductive changes.  Inasmuch  as  bacteria  possess  the  power 
of  multiplication,  it  might  be  expected  that  they  would 
contain,  like  higher  forms,  a  well  denned  nuclear  body. 

The  direct  examination  of  a  bacterial  cell  fails  to  reveal 
the  presence  of  any  form  analogous  to  a  nucleus.  Usually, 
the  cell  appears  perfectly  homogeneous,  and  the  closest 
examination  will  not  bring  out  a  structure.  The  question 
-of  the  existence  of  nuclei  in  bacteria  has  been  the  subject 
of  numerous  extensive  investigations  which  have  led  to 
very  different  conclusions.  In  fact,  it  may  be  said  that  the 
presence  of  a  nucleus  is  not  demonstrated. 

There  are  some  who  consider  bacteria  as  wholly  devoid 
-of  nuclei.  The  minute  granules  which  are  not  infrequently 
present,  especially  in  older  cells,  are  believed  by  some  to 
be  the  first  indication  of  the  formation  of  a  nucleus. 

Some  have  endeavored  to  show  the  existence  of  a  cen- 
tral body  which,  while  it  is  not  a  nucleus  in  the  sense  that 
this  term  is  usually  employed,  nevertheless  possesses  certain 
properties  of  a  nucleus,  and  may  therefore  be  considered  as 
-a  rudimentary  form  of  that  body.  On  the  whole  the  opinion 
prevails  that  bacteria  consist  essentially  of  nuclear  matter 
surrounded  by  a  very  thin  protoplasmic  layer  and  by  the 
cell-wall.  It  is  well  known  that  in  embryonic  cells  the 
nucleus  almost  fills  the  cell. 

This  view  is  especially  strengthened  by  the  behavior 
of  bacteria  to  anilin  dyes.  The  bacterial  cells  stain  readily 
and  intensely,  like  the  nuclei  of  higher  cells.  This  is 
assumed  to  be  due  to  the  presence  of  similar  chemical  sub- 
stances such  as  nucleins.  The  presence  of  a  nuclein  com- 
pound in  the  bacterial  cell  is  indicated  by  the  fact  that 
nuclein  bases  and  even  a  protamin-like  body  have  been 
isolated. 


32  BACTERIOLOGY. 

As  stated  above,  the  cellular  contents  of  most  bacteria 
appear  perfectly  homogenous.  Certain  species,  however, 
show  the  presence  of  various  sized  granules.  These  are 
especially  present  in  old  cultures  and  seem  to  have  a  protein 
composition.  They  may  be  related  to  the  chromatin  of 
higher  cells  and,  as  pointed  out  above,  they  are  believed  by 
some  to  represent  the  earliest  form  in  the  development  of  a. 
nuclear  body.  These  granules  may  be  large  or  small,  very 
numerous  or  apparently  absent.  It  is  quite  possible  that 
these  are  due  to  condensation  of  the  protoplasm  as  a  result 
of  plasmolytic  changes.  Similar  bodies  appear  in  the  cell 
contents  invariably  previous  to  spore  formation,  and  these 
have  been  termed  sporogenic  granules. 

In  the  process  of  drying,  fixing  and  staining  bacteriav 
artificial  changes  not  infrequently  occur  which  may  be  con- 
sidered as  evidence  of  a  structure  which  in  reality  does  not 
exist.  For  instance,  a  bacterial  suspension  is  allowed  to  dry 
on  a  cover  glass.  As  the  water  evaporates  the  concentration 
of  the  salts  present  is  increased,  and  as  a  result  marked  plas- 
molytic changes  may  result.  The  cell  may  then  show  marked 
granules  or  polar  bodies  and  vacuoles.  The  socalled  spores 
of  the  tubercle  bacillus  may  be  due  to  changes  of  this  kind. 

Certain  bacteria,  such  as  the  typhoid  and  chicken 
cholera  group,  on  feeble  staining  show  well  stained  ends 
separated  by  an  almost  colorless  zone.  This  is  spoken  of 
as  the  bi-polar  stain,  and  is  supposed  to  be  due  to  the  pres- 
ence of  a  vacuole  or  cell  fluid  in  the  middle  of  the  cell. 
The  protoplasm  is  consequently  pushed  to  each  side  and, 
owing  to  its  dense  condition,  readily  takes  up  the  stain. 
This  may  represent  the  normal  conditions  of  the  cell  but, 
on  the  other  hand,  it  may  be  due  to  the  plasmolytic  changes 
mentioned  above.  These  polar  bodies  are  certainly  not 
spores,  as  has  been  at  one  time  supposed. 

The  contents  of  the  bacterial  cell  are,  as  a  rule,  per- 
fectly colorless.  This  is  true  even  though  the  organism 


SIZE  AND  STRUCTURE  OF  THE  BACTERIAL  CELL.  33 

produces  a  brilliant  pigment.  In  this  case  the  pigment,  or 
rather  its  antecedent,  is  made  within  the  cell  and  is  then 
excreted  into  the  surrounding  medium. 

A  few  bacteria  show  a  faint  red  coloration  when  exam- 
ined under  the  microscope.  This  is  due  to  the  presence  of 
a  red  pigment  known  as  bacterio-purpurin.  This  substance, 
in  its  microchemical  reactions,  appears  to  be  identical  with 
a  similar  coloring  matter  present  in  certain  protozoa.  It  is 
believed  to  exercise  a  role  similar  to  that  of  chlorophyll. 

A  green  chlorophyll-like  pigment  has  been  observed  to 
be  present  in  a  small  number  of  bacteria.  The  absence  of 
chlorophyll  from  the  majority  indicates  that  these  organ- 
isms, unlike  the  higher  plants,  cannot  assimilate  carbonic 
acid.  This,  however,  does  not  necessarily  follow,  since 
certain  nitrifying  bacteria  are  capable  of  assimilating  car- 
bonic acid  even  in  the  absence  of  light. 

A  number  of  bacteria  have  been  met  with  in  the  mouth 
which  show  a  yellowish  or  brownish  tint.  The  exact  nature 
of  the  pigment  in  either  of  these  cases  is  unknown. 

On  contact  with  iodine,  protoplasm  in  general  takes  on 
a  light  yellow  color.  A  number  of  bacteria,  however,  give 
with  iodine  a  blue  or  dark  violet  color.  Inasmuch  as  this 
reaction  is  similar  to  that  with  boiled  starch  or  granulose, 
it  is  spoken  of  as  the  granulose  reaction.  Such  bacteria 
would  seem  to  contain,  therefore,  a  carbohydrate  similar  to 
soluble  starch.  This  substance  may  be  present  only  in 
scattered  granules,  or  these  may  accumulate  so  that  the 
entire  cell  is  deeply  stained.  In  some  bacteria  the  substance 
is  not  present  in  the  cell  until  just  previous  to  spore  forma- 
tion. Bacteria  giving  this  reaction  are  especially  found  in 
the  mouth. 

Motility  of  bacteria.  When  bacteria  are  examined  in  the 
living  condition  they  will,  as  a  rule,  show  motion.  The 
movements  observed  may  be  apparent  or  they  may  be  real. 
In  the  latter  case  the  organism  is  in  active  motion,  travel- 

3 


34  BACTERIOLOGY. 

ing  from  one  place  to  another.  The  impulse  that  creates 
this  real,  active  motion  comes  from  within  the  cell,  in 
other  words  from  the  living1  protoplasm.  On  the  other 
hand,  many  bacteria  will  show  an  apparent  motion.  The 
cells  move  to  and  fro,  trembling  as  it  were,  but  do  not 
actually  change  their  relative  position.  The  impulse  in 
this  case  comes  from  without  the  cell,  which  itself  remains 
passive. 

The  apparent  motility  of  bacteria  is  known  as  Brownian, 
or  molecular,  or  physical  motion.  All  exceedingly  minute 
objects,  when  suspended  in  the  air  or  in  a  fluid,  will  show 
this  peculiar  swaying  or  pendulum  movement.  The  mole- 
cules composing  the  air  or  fluid  are  in  constant  motion,  and 
consequently  strike,  again  and  again,  the  minute  objects 
that  may  be  in  their  path.  This  molecular  bombardment 
of  an  otherwise  motionless  bacterial  cell  causes  it  to  sway 
to  and  fro. 

It  may  at  times  be  very  difficult  to  distinguish  between 
Brownian  and  real  motion.  In  that  case  it  is  necessary 
to  resort  to  certain  experiments.  Brownian  motion  is 
obviously  manifested  by  dead  as  well  as  by  living  cells. 
The  organism  may  be  destroyed  by  heating  at  60°  for  one 
hour,  or  it  may  be  treated  with  a  germicidal  substance  such 
as  carbolic  acid,  or  mercuric  chloride.  If  the  dead  cell 
exhibits  the  same  motion  as  the  living  cell,  it  is  clearly  due 
to  purely  physical  causes.  Again,  by  growing  the  organism 
at  a  constant  low  temperature  of  15°  or  less  it  will  in  many 
cases  become  larger  than  usual,  and  consequently  will 
respond  less  readily  to  the  impact  of  molecules.  This  pro- 
cedure is  especially  useful  in  doubtful  cases.  Finally,  as  a 
rule,  it  is  possible  to  distinguish  between  the  two  kinds  of 
motion  by  demonstrating  the  presence  or  absence  of  the 
characteristic  organs  of  motion.  If  these  are  present  there 
can  be  no  doubt  of  the  true  motility  of  the  organism.  On 
the  other  hand,  failure  to  find  these  motile  organs  does  not 
prove  that  they  are  absent,  inasmuch  as  in  some  undoubt- 


SIZE  AND  STRUCTURE  OF  THE  BACTERIAL  CELL.  o5 

edly  motile  bacteria  it  is  very  difficult  to  demonstrate  their 
presence. 

Bacteria  exhibit  real  motion  only  in  liquid  or  on  moist 
media,  and  then  only  when  in  the  actively  growing  con- 
dition. When  in  the  seed  or  spore  form  they  do  not  possess 
motion;  neither  do  they  possess  active  motion  when  floating 
about  in  the  air  as  fine  dust-like  particles. 

Real  motion  is  observed  in  most  of  the  spiral-shaped 
bacteria.  The  bacilli  are,  as  a  rule,  motile.  The  micro- 
cocci  are,  on  the  other  hand,  usually  non-motile.  Two  or 
three  micrococci  are  known  to  possess  motion.  Two  motile 
sarcines  have  also  been  studied. 

The  motion  exhibited  by  bacteria  will  vary  with  the 
different  species,  and  probably  depends  upon  the  number 
and  arrangement  of  the  organs  of  locomotion.  Some  rods 
show  a  slow,  forward,  wabbling  movement.  Others  glide 
rapidly  and  steadily  forward.  In  some  the  forward  motion 
is  accompanied  by  a  rotation  of  the  cell  around  its  long 
axis,  while  in  others  a  "somersault"  movement  is  to  be 
seen.  Frequently  the  actively  motile  cell  will  suddenly 
reverse  its  motion  and  travel  backward.  It  may  come  to  a 
sudden  stop,  and  then  as  suddenly,  again  begin  to  move. 

The  motility  of  a  culture  depends  largely  upon  its  age, 
upon  the  composition  of  the  soil,  and  upon  the  temperature. 
In  the  case  of  anaerobic  bacteria  the  presence  of  oxygen 
soon  inhibits  motion.  The  higher  the  temperature,  as  a 
rule,  the  more  marked  will  be  the  movement.  Thus,  certain 
bacteria  scarcely  show  real  motion  at  the  ordinary  room 
temperature  because  of  the  presence  of  a  slimy  secretion. 
When  placed,  however,  at  the  temperature  of  the  body  the 
motion  becomes  well  marked. 

Active  motion  is  carried  on  by  means  of  certain  organs 
known  a.s-flagella  or  whips.  The  whips  are  very  delicate, 
long,  thin  threads  of  protoplasmic  substance.  They  are 
analogous  to  similar  appendages  on  motile  infusoria,  on 
plants  and  on  ciliated  epithelium.  Owing  to  their  extreme 


36 


BACTERIOLOGY. 


delicacy  they  can  only  very  rarely  be  seen  in  the  living 
condition.  Moreover,  they  do  not  color  with  anilin  dyes  in 
the  same  way  as  the  mass  of  the  bacterial  cell,  and  for  that 
reason  they  are  not  visible  in  the  ordinary  stained  prepara- 
tion. In  order  to  demonstrate  the  presence  of  flagella  or 
whips  it  is  necessary  to  resort  to  a  very  special  method  of 
staining1.  They  then  appear  as  slender,  wavy  filaments. 
It  is  the  lashing  of  these  whips  that  propels  the  organism 
through  the  liquid. 


FIG.  6.  Motile  organs  or  whips  on  bacteria,  a— Microcpccus;  ^—Bacillus  with  term- 
inal whips;  c — Bacillus  with  diffuse  whips;  d — Vibrio;  e — Single  spirillum  with  bunch  of 
whips;  /—Spirillum  after  division  with  whips  at  each  end. 

The  flagella  will  vary  considerably  in  size  in  different 
species,  and  even  at  times  in  individuals  of  the  same  spe- 
cies. Their  width  is  usually  less  than  ^  of  the  width  of 
of  the  cell.  Their  length  will  usually  be  two  or  three  times 
the  length  of  the  cell,  but  it  is  not  uncommon  to  find  some 
bacteria  with  whips  that  are  ten  or  twenty  times  as  long  as 
the  cell. 

The  flagella  project  from  the  outer  border  of  the  organ- 
ism. They  are  supposed  by  some  to  be  directly  continuous 
with  the  protoplasm  within  the  cell.  In  that  case  the 
protoplasmic  threads  are  supposed  to  pass  through  minute 
openings  in  the  cell-wall.  Plasmolytic  experiments,  how- 
ever, do  not  indicate  a  protoplasmic  continuity.  '  On 
treatment  with  saline  solutions,  as  indicated  above,  the 
protoplasm  of  the  living  cell  withdraws  completely  from 
the  cell-wall  and  gathers  in  one  or  two  round  masses.  If 


SIZE  AND  STRUCTURE  OF  THE  BACTERIAL  CELL.  37 

the  flagella  are  merely  projecting  threads  of  protoplasm  it 
might  be  expected  that  in  plasmolysis  they  would  be  with 
drawn  within  the  cell  and  that  motion  would  cease.  This, 
however,  is  not  the  case.  The  plasmolyzed  living  cell 
continues  to  move  the  same  as  in  the  beginning.  The 
flagella,  therefore,  are  not  directly  connected  with  the  inner 
protoplasm  of  the  cell.  They  are  given  off  by  the  cell-wall 
and  chemically  they  would  seem  to  be  identical  with  the 
outer,  softened  layer  of  this  structure. 

As  indicated  above,  the  cell-wall  is  essentially  protein 
matter  and,  unlike  the  cellulose  wall  of  higher  plant  cells, 
it  takes  an  active  part  in  the  life  of  the  cell.  The  cell-wall 
receives  its  nourishment  from  the  protoplasm  and  is  itself, 
therefore,  a  living  structure.  The  filaments  or  whips  given 
off  by  the  outer  layer  of  this  wall  are  also  protein  in  nature 
and  are  also  living.  The  flagella  are  unquestionably  the 
organs  of  motion.  By  inducing  movements  in  the  liquid 
they  renew  continually  the  food  supply.  Moreover,  it  is 
possible  that  the  protoplasmic  whip  is  a  means  of  absorb- 
ing nourishment  for  the  cell.  In  the  latter  case  it  might  be 
expected  that  flagella  would  also  be  present  on  non-motile 
bacteria.  Flagella,  however,  are  never  found  on  strictly 
non-motile  organisms.  Pseudo-flagella,  due  to  a  mucous- 
like  secretion,  are  sometimes  met  with  in  such  cases. 

The  arrangement  of  flagella  on  a  given  species  is  fairly 
constant,  but  will  vary  with  different  species.  In  the  vibrio 
of  Asiatic  cholera,  and  in  the  bacillus  of  green  pus,  there  is 
usually  only  one  whip  present  and  that  is  attached  to  one 
end.  At  times,  the  cholera  vibrio  may  have  two,  three  or 
four  whips  at  one  pole,  and  again  it  may  have  none.  In 
old  cultures  it  may  have  a  whip  at  each  end  (Fig.  6  d). 

The  spiral  forms,  as  a  rule,  have  a  bunch  of  whips  at 
one  end.  When  each  end  is  equipped  with  a  bunch  of 
whips  it  is  because  there  are  really  two  cells  present.  The 
whips  on  the  spiral  forms  are  not  as  flexible  and  wavy  as 
those  on  the  bacilli.  They  appear  rather  stiff  and  are 


38  BACTERIOLOGY. 

slightly  curved,  like  an  eye-lash.  They  can  therefore  be 
designated  as  cilia  rather  than  as  flagella  (Fig.  6  e,  /). 

In  many  bacilli  the  flagella  are  very  numerous,  and  are 
diffuse,  or  distributed  all  over  the  surface  of  the  cell.  In 
such  cases  a  perfect  fringe  of  delicate  wavy  lines  can  be 
seen  surrounding  the  organism.  The  Proteus  vulgaris, 
typhoid  bacillus  and  the  anaerobic  bacteria  are  especially 
well  provided  with  flagella  (Fig.  6  c). 

Flagella  are  especially  abundant  on  fresh  young 
growths,  about  one  day  old.  They  disappear  in  old  cul- 
tures by  being  torn  off.  Usually,  they  also  disappear  just 
before  spore  formation.  This,  however,  is  not  the  case 
with  anaerobic  bacteria.  The  formation  of  whips  depends 
upon  the  composition  of  the  soil.  Thus,  cultures  of  the 
cholera  and  typhoid  bacilli  which  have  been  grown  on  the 
ordinary  artificial  media  by  the  author  for  more  than  ten 
years  show  scarcely  any  motion.  It  has  been  proposed  to 
employ  the  fact  of  the  number  and  arrangement  of  whips 
as  a  means  of  identification  of  species,  but  for  reasons  indi- 
cated above  this  is  not  feasible. 

In  1890  Loffler  observed  in  stained  preparations  and  in 
a  living  culture  of  the  bacillus  of  symptomatic  anthrax 
enormous  spindle-shaped  spiral  bodies,  which  he  believed 
to  be  formed  like  a  braid  of  hair,  by  the  twisting  together 
of  a  large  number  of  the  ordinary  whips.  Three  years  later 
the  author  met  with  these  same  *  'giant-whips  "  while  study- 
ing a  new  anaerobic  bacillus,  and  was  also  able  to  repeat- 
edly demonstrate  their  presence  in  three  other  anaerobic 
bacteria.  In  the  same  year  Sakharoff  met  with  these  pecu- 
liar forms  in  gelatin  cultures  of  the  B.  Asiaticus.  Recently, 
A.  Fischer  has  described  giant-whips  in  several  species,  and 
Sames  has  found  the  same  forms  in  cultures  of  a  motile 
sarcine.  The  author,  during  the  past  year,  has  studied  the 
development  of  these  enormous  spirals  in  cultures  of  the 
typhoid,  coli,  psittacosis,  and  icteroides  bacilli.  There  is 


SIZE  AND  STRUCTURE  OF  THE  BACTERIAL  CELL.  39 

reason,  therefore,  to  believe  that  all  motile  bacteria  give 
rise  to  these  so-called  giant-whips.  It  is  possible  that  the 
spirals  observed  in  the  intestinal  contents  of  cholera,  and 
those  present  in  hospital  gangrene,  are  not  distinct  organ- 
isms, but  rather  altered  flagella.  The  author  has  found 
giant  whips  in  the  bodies  of  animals  (Fig  7  a)1. 

Their  size  can  be  inferred  from  the  fact  that  they  can 
be  seen  in  unstained  specimens.  The  larger  forms  can  be 
easily  seen  with  a  No.  3  objective.  The  author  has  repeat- 
edly found  giant-whips  that  were  70  p.  in  length.  In  one 
instance  the  length  was  132  ,u,  or  rie  of  an  inch. 


FIG.  7.  Giant  whips,  a  and  b  from  photographs  of  Bacillus  oedematis  maligni 
No.  II.  a — Colorless  spiral  in  streak  preparation  from  peritoneum  of  a  guinea-pig — 
bacilli  stained;  b — Large  spindle-shaped  spiral,  compared  with  ordinary  whips;  c — 
Slender,  long  spiral  form. 

The  giant-whips  are  invariably  motionless  and  usually 
are  spindle-shaped.  In  this  case  the  borders  are  wavy  and 
corresponding  diagonal  bands  will  be  seen,  resembling  the 
twisted  appearance  of  a  rope.  Sometimes  a  spindle  seems 
to  divide  lengthwise,  so  that  it  appears  as  if  two  spindles 
diverged  from  a  common  point.  The  thick  spindle  is  not 
the  only  form  in  which  the  giant-whip  is  met  with.  It  may 
be  a  slender,  wavy,  very  long  spiral,  without  any  enlarge- 
ment or  thickening.  Such  spirals  may  extend  through  the 
entire  field.  These  thread-shaped  giant-whips  may  be 
single,  but  they  may  also  be  bunched,  proceeding,  as  it 
were,  from  a  common  point.  The  giant-whips  are  especially 
abundant  in  the  water  of  condensation  which  is  present  in 

1  Zeitschrift  fur  Hygiene,  17,  plates  1  and  2. 


40  BACTERIOLOGY. 

a   tube   of   freshly   inclined   agar.     The   method   for  their 
detection  will  be  given  in  Chapter  XI. 

As  mentioned  above,  Loffler,  the  discoverer  of  these 
strange  forms,  considered  them  to  be  woven  masses  of 
the  ordinary  whips,  and  this  view  has  been  quite  generally 
accepted.  It  is  doubtful,  however,  that  this  explana- 
tion of  their  origin  is  correct.  If  they  result  from  a  twist- 
ing process  some  motion  should  at  times  be  observable, 
but  such  is  not  the  case.  The  author  has  observed  beauti- 
ful small  spindles  in  cultures  only  eight  hours  old,  but  at 
no  time  could  motion  be  observed.  Moreover,  the  spindles, 
especially  in  the  earliest  stage,  might  be  expected  to  be 
surrounded  by  the  bacteria  which  have  had  their  whips 
entangled.  Usually,  however,  the  whips  stand  out  sharp 
by  themselves.  Furthermore,  the  braiding  process  does 
not  satisfactorily  explain  the  formation  of  the  perfectly 
even  and  very  thin  spirals  which  frequently  attain  a  length 
of  100  P.  or  more.  It  has  been  supposed  by  some  that  the 
ordinary,  flagella,  which,  as  pointed  out,  may  be  considered 
as  living  protoplasmic  matter,  when  torn  loose  from  the 
cell,  may  continue  to  move  about  for  a  short  time,  and  in 
this  way  lead  to  the  formation  of  giant-whips.  There  is  rea- 
son to  believe  that  the  motile  organs  on  certain  flagellates, 
or  animal  organisms,  are  endowed  with  contractility  and, 
for  a  short  time  at  least,  may  live  and  move  about  after 
separation  from  the  cell  proper.  It  is  possible,  however, 
that  these  forms  are  unusually  developed  flagella,  either 
as  a  result  of  involution  changes,  or  because  of  a  softening 
of  the  whip  substance,  corresponding  to  that  observed  in 
capsule  formation. 


CHAPTER  III. 
THE  LIFE  HISTORY  OF  BACTERIA. 

Growth  and  multiplication  is  a  characteristic  of  living 
organisms.  As  a  rule  the  plant  or  animal  cell,  when  it 
reaches  the  fully  developed,  adult  stage,  divides  and  thus 
gives  rise  to  two  new  cells.  The  young  bacterial  cell,  like- 
wise, grows,  attains  its  full  size,  and  then  multiplies  by 
division  or  fission.  Bacteria  are,  for  this  reason,  designated 
as  schizomycetes  or  fission-fungi. 

The  multiplication,  or  actual  increase  in  number,  of 
bacteria  always  results  by  the  process  of  division  whereby 
one  cell  forms  two,  and  only  two,  new  cells.  There  are 
instances,  as  will  presently  be  seen,  where  apparently  one 
cell  gives  rise  to  four  or  to  eight  cells,  but  in  all  such  cases 
the  division  is  consecutive  and  not  direct.  That  is  to  say, 
the  cell  does  not  divide  directly  into  fourths  or  eighths,  but 
does  form  two  halves  which,  subsequently  dividing,  yield 
four  cells,  and  the  next  division  yields  eight  cells. 

Cell-division  among  uni-cellular  plants  and  animals  is 
completed  in  a  very  short  length  of  time.  This,  perhaps, 
is  especially  true  of  the  bacteria.  Given  a  suitable  soil, 
the  rapidity  of  growth  and  multiplication  of  bacteria  will 
depend  upon  the  temperature.  The  nearer  the  temperature 
approaches  the  freezing  point  the  slower  will  be  the  rate  of 
multiplication.  On  the  other  hand  a  temperature  of  30°  to 
37°  gives  the  most  rapid  growth.  Under  such  conditions 
the  average  bacterial  cell  will  probably  divide  in  less  than 
a  half  an  hour.  This  rate  of  multiplication  cannot  be  main- 
tained for  any  length  of  time,  owing  to  the  exhaustion  of  the 
soil  and  above  all  to  the  accumulation  of  waste  products 


42  BACTERIOLOGY. 

which  retard  and  eventually  stop  growth.  Assuming-  the 
continuance  of  favorable  conditions,  a  single  cell,  dividing 
in  30  minutes,  would  be  represented  at  the  end  of  12  hours 
by  more  than  sixteen  million  descendants.  In  24  hours  the 
number  would  rise  to  more  than  two  hundred  and  eighty 
billions. 

The  above  figures  will  serve  to  impress  the  fact  that 
bacteria  multiply  with  extreme  rapidity.  Moreover,  they 
will  help  to  understand  what  marked  chang-es  may  result  in 
a  comparatively  short  period  of  time  when  certain  bacteria 
develop  in  milk,  meats  or  in  the  living-  animal  body.  Small 
as  bacteria  are,  owing-  to  their  rapid  multiplication,  they 
may  give  rise  to  poisonous  substances  in  sufficient  amount 
to  cause  in  a  few  hours  profound  poisoning,  or  even  death. 


FIG.  8. — Cell  division  (diagrammatic),    a — Bacillus,  showing  deflection  of  outer 
membrane;    b— Micrococcus. 

The  process  of  division  can  best  be  observed  in  large 
bacilli.  Owing  to  the  absence  of  a  definite  nucleus,  and  of 
any  cell  structure,  the  change  that  takes  place  during 
multiplication  is  very  simple.  When  the  bacillus  has 
attained  the  fully  developed  stage,  a  slight  transverse 
constriction  appears  at  the  middle  of  the  rod.  The  ring- 
like  process  of  the  cell-wall  gradually  extends  toward  the 
center  of  the  cell  until  eventually  the  protoplasm  becomes 
divided  into  two  halves.  Usually  the  first  indication  that 
cell  division  has  taken  place  is  the  appearance  of  the  clear, 
delicate  transverse  line.  The  division  of  bacilli  and  of 
spirals  is  always  transverse,  never  longitudinal. 

The  wall  that  divides  the  cell  into  two  is  to  be  consid- 
ered as  an  ingrowth  of  the  cell  membrane.  Inasmuch  as  in 


THE   LIFE  HISTORY  OF    BACTERIA.  43 

stained  preparations  this  dividing  line  remains  colorless,  it 
is  evident  that  it  largely  consists  of  the  same  material  as 
the  outer  layer  of  the  cell  membrane,  namely,  the  softened, 
gelatinized  mantle  which,  unlike  the  membrane  proper, 
does  not  stain  with  anilin  dyes.  Obviously  the  entire  cell- 
wall,  inner  as  well  as  outer  membrane,  is  depressed  at  the 
zone  of  constriction  (Fig.  8  a).  When  the  ingrowing  wall 
reaches  the  center  the  inner  membrane  coalesces  and 
divides,  leaving  the  space  between  the  two  new  end  walls 
filled  with  the  gelatinized  outer  membrane.  Continuous 
filaments  showing  no  apparent  division  into  cells  are  some- 
times met  with.  In  such  cases  division  may  have  occurred, 
but  is  not  visible,  owing  to  the  absence  of  the  outer  mem- 
brane. 

/ 


FIG.  9.    Division  forms  of  bacilli,    a — single;    b — in  pairs;    c — in  threads. 

As  soon  as  the  cell  has  completely  divided,  the  two 
new  cells  may  at  once  tear  apart  and  lead  a  separate  exist- 
ence. Many  bacilli  are  therefore  characterized  as  growing 
singly.  In  some  species  there  is  a  tendency  for  the  two 
cells  to  remain  attached  owing  to  the  firm  union  of  the  two 
cells,  which  are  held  together  by  the  gelatinous  connecting 
zone.  The  bacillus  is  then  spoken  of  as  growing  in  pairs — 
diplo-bacillus.  In  other  species  the  cells  are  likely  to  remain 
attached  even  after  repeated  division.  The  individual  rods 
remain  attached,  end  to  end,  by  the  undivided  outer  layer 
of  the  cell-wall,  and  thus  give  rise  to  long  filaments  which 
are  commonly  designated  as  threads.  A  thread  may  be  long 
or  short;  that  is,  it  may  consist  of  four  or  five,  or  fifty  to  one 


44  BACTERIOLOGY. 

hundred  or  more  cells.  A  thread  is  always  composed  of 
rod-shaped  bacteria,  bacilli,  and  may  be  compared  to  a  row 
of  bricks  in  a  wall. 

Micrococci  multiply  in  like  manner  by  division.  The 
spherical  organism  is  usually  supposed  to  elongate  some- 
what just  previous  to  fission  (Fig".  8  &).  The  transverse 
constriction  and  dividing-  line  then  appear  as  in  the  case  of 
a  bacillus.  According-  to  some  the  micrococcus  does  not 
elongate  first,  but  divides  directly  into  two  halves.  It  may 
increase  in  size  previous  to  division,  but  its  form  remains 
spherical.  On  the  other  hand  the  bacillus  always  grows  in 
length,  and  it  would  seem  as  if  this  fact  could  be  utilized 
in  distinguishing-  between  a  true  micrococcus  and  a  very 
short  rod. 


oo 
go 


FIG.  10.  Division  forms  of  micrococci.  a — diplococcus,  perfect  form — with 
flattened  apposed  surface  (gonococcus),— lanceolate  form  (pneumonia);  ^—strepto- 
coccus; c — Consecutive  division  yielding  a  tetrad;  d — Sarcine  form  resulting  from 
division  of  tetrad  c.  e— Staphylococcus. 

The  two  half-spheres  which  result  from  the  division  of 
micrococcus  may  retain  this  form  and  remain  attached  by 
the  undivided  cell-wall.  In  this  case  the  two  cells  are 
spoken  of  as  being  biscuit-shaped  and  in  pairs.  The  germ 
of  gonorrhea  presents  this  characteristic  appearance. 
When  micrococci  grow  in  pairs,  as  in  this  instance,  they 
are  designated  as  diplococci  (Fig.  10  a). 

On  the  other  hand,  as  soon  as  the  division  is  complete, 
the  two  new  cells  may  gradually  round  out  and  assume  a 
spherical  form.  They  may  tear  apart  and  grow  singly;  or, 
the  two  cells  may  remain  attached  by  the  narrow  zone  of 
undivided  cell-wall,  forming  thus  a  diplococcus.  If  each  of 
these  two  attached  cells  now  divides  in  the  same  direction 
as  the  original  one,  a  row  of  four  spherical  organisms  will 
result.  A  continuation  of  this  division  in  one  direction 


THE  LIFE   HISTORY  OF   BACTERIA.  45 

eventually  yields  a  long  row  or  chain  of  attached  micro- 
cocci.  This  exceedingly  characteristic  form  is  known  as  a 
streptococcus,  and  as  indicated,  its  formation  is  analogous  to 
that  of  a  thread.  It  may  be  compared  to  the  beads  on  a 
rosary  (Fig.  10  &). 

In  the  above  instances  the  dividing  membrane  always 
forms  in  a  plane  parallel  to  the  original  plane  of  division.- 
Consequently  growth  extends,  as  it  were,  along  a  line.  The 
division  is  then  said  to  occur  in  one  direction  of  space. 
This  does  not  hold  true  for  all  micrococci.  Thus,  after  the 
original  cell  divides  into  two,  each  of  these  in  turn  may 
divide  so  that  the  line  of  division  is  at  right  angles  to  that 
in  the  original  cell.  The  four  cells  which  thus  result  are 
not  arranged  in  a  row,  but  form  a  tetrad,  which  may  be  com- 
pared, when  stained,  to  the  four  spots  on  a  die.  Division 
has  been  consecutive  and  in  two  directions  of  space  (Fig. 
10  c;  also  Fig.  5  &). 

Again,  each  of  the  four  cells  of  a  tetrad  may  divide  so 
that  the  plane  of  division  will  be  parallel  to  the  face  of  the 
tetrad.  The  result  is  a  cubical  mass  of  eight  cells.  This 
package-shaped  mass  of  micrococci  is  known  as  a  sarcine. 
Division  has  been  consecutive  and  in  three  directions  of 
space  (Fig.  10  d). 

Lastly,  micrococci  may  divide  rather  irregularly  and, 
remaining  adherent,  eventually  yield  a  mass  of  cells  which 
resemble  somewhat  a  bunch  of  grapes.  Such  forms  are 
designated  as  staphylococci  (Fig.  10  e). 

Whatever  may  be  the  final  form  which  results  from  the 
division  of  micrococci,  whether  the  division  occurs  in  one, 
two  or  three  directions  of  space,  it  is  always  to  be  under- 
stood as  consecutive.  That  is  to  say,  one  cell  divides  into 
two;  these  two,  on  division,  yield  four  cells,  and  the  latter 
in  turn  yield  eight  cells.  One  micrococcus  never  divides 
directly  into  quarters  or  into  eighths. 

The  simple  micrococcus,  as  shown  above,  may  multiply 
so  that  it  usually  appears  as  a  single  cell.  When  the  cells 


46  BACTERIOLOGY. 

remain  attached  because  of  the  incompletely  divided  cell- 
wall,  diplococci,  streptococci,  tetrads,  sarcines  and  staphy- 
lococci  may  result.  As  a  rule,  a  given  micrococcus  shows 
some  one  of  these  six  forms  as  its  characteristic  form. 
Thus,  a  micrococcus  that  forms  a  streptococcus  will  not 
occur  as  a  tetrad  or  as  a  sarcine. 


FIG.  ii.    Division  forms  of  the  spirillum,    a— Vibrio,  b— 
cell,  form  of  elongated  S;     c — Long  spirillum,  showing  compraa  , 

cells. 

The  spiral  or  screw-shaped  bacteria  multiply  by  trans- 
verse division,  as  in  the  case  of  bacilli.  When  the  organ- 
ism grows  single  it  forms  a  bent  and  twisted  rod  which,  as 
ordinarily  seen,  appears  comma-shaped.  The  comma 
bacilli,  or  vibrios,  are  the  individual  cells  which,  when  they 
remain  attached,  end  to  end,  yield  a  spirillum  (Fig-.  11). 
The  spirillum,-  therefore,  may  be  considered  as  analogous 
to  the  thread,  and  streptococcus.  Certain  spirilla,  like 
those  of  the  mouth  and  of  recurrent  fever,  cannot  be 
resolved  into,  component  cells,  and  are  therefore  to  be 
considered  as  single  individuals. 


Spores. 

It  has  been  pointed  out  above  that  bacteria  always 
multiply  by  division.  In  this  process  one  cell,  dividing 
into  two  halves,  yields  two  new  individuals.  As  long-  as 
the  organism  is  growing  and  multiplying  it  is  said  to  be 
vegetating  and  the  form  which  it  presents  during  this 
period  of  its  life  is  spoken  of  as  the  vegetative  form. 
This  stage,  therefore,  may  be  compared  to  the  growing, 
higher  plant.  The  latter,  however,  when  it  reaches  the 
adult  condition  develops  reproductive  organs  and  forms 
seeds. 


THE  LIFE  HISTORY  OF    BACTERIA.  47 

A  somewhat  similar  condition  is  observed  among-  many 
bacteria.  The  active  growth  stops  and  reproduction  occurs. 
The  organism  in  this  case  gives  rise  to  what  is  called  a 
spore  which  is  the  analogue  of  the  seed  of  a  higher  plant. 
The  object  of  either  seed  or  spore  formation  maybe  said 
to  be  the  perpetuation  of  the  species.  The  plant,  whether 
high  or  low,  when  in  the  vegetating  condition  is  a  relatively- 
weak  organism.  It  is  readily  destroyed  by  desiccation, 
heat,  cold  and  other  agencies.  There  is  need,  therefore, 
of  some  resistant  form  which  will  enable  the  plant  to  sur- 
vive unfavorable  external  conditions.  The  flowering  plant 
forms  its  seed,  whereas  the  flowerless  plant  forms  the  spore. 

It  is  evident,  therefore,  that  bacteria  may  be  either  in 
the  actively  growing,  vegetating  form,  or  in  the  reproductive 
or  spore  form.  The  latter  is  sometimes  spoken  of  as  the 
resting,  permanent  or  resistant  form.  As  will  presently  be 
shown  the  bacillus,  when  it  sporulates  or  forms  spores, 
almost  invariably  gives  rise  to  a  single  spore.  The  latter, 
in  turn,  when  it  sprouts  or  germinates  gives  rise  to  a  single 
bacillus.  Consequently  it  is  not  proper  to  speak  of  bacteria 
as  multiplying  by  spore  formation.  When  the  parent  cell 
gives  rise  to  but  one  spore  which  in  turn  develops  into  one 
young  cell  the  process  cannot  be  considered  as  a  mul- 
tiplication or  an  increase  of  species.  Bacteria  reproduce, 
perpetuate  themselves  by  means  of  spores  but  they  multiply 
by  cell  division.  The  vegetating  cell  may  divide,  or  may 
form  a  spore,  whereas  the  spore  can  only  germinate. 

The  formation  of  spores  has  been  observed  in  some 
spirals,  in  many  bacilli  but  not  among  micrococci.  The 
vast  majority  of  bacteria,  therefore,  have  not  been  seen  to 
possess  spores.  From  this  it  by  no  means  follows  that 
these  bacteria  always  remain  sporeless.  The  conditions  of 
spore  formation  are  as  yet  but  imperfectly  understood  and 
it  is  quite  probable  that  many,  if  not  all,  of  the  bacteria  in 
which  spores  have  not  been  found  can  give  rise  to  these 
bodies  under  the  conditions  which  prevail  in  nature.  The 


48  BACTERIOLOGY. 

conditions  under  which  bacteria  exist  in  the  laboratory  are 
far  from  being"  the  best. 

The  spore  is  always  formed  within  the  cell.  It  may 
therefore  be  spoken  of  as  an  endospore.  The  botanist 
DeBary  attempted  to  make  use  of  this  fact  as  the  basis  of 
a  natural  classification  of  bacteria.  He  divided  the  group 
into  endospore  and  arthrospore  bacteria.  In  arthrospore 
formation  it  was  supposed  that  the  entire  cell  converted 
itself  into  a  spore-like  resisting  form.  That  is  to  say,  the 
cell-wall  of  the  individual  organism  would  thicken,  harden 
and  become  impenetrable.  In  this  condition  the  cell,  like 
a  spore,  could  survive  unfavorable  conditions  and  could  in 
turn  germinate  or  multiply.  All  bacteria  which  were  not 
known  to  produce  endospores  were  therefore  placed  in  the 
arthrospore  group.  As  a  matter  of  fact  there  is  no  good 
and  sufficient  reason  to  believe  in  the  existence  of  arthro- 
spore formation. 

Sporulation. — Owing  to  the  extremely  small  size  of 
bacteria  it  is  manifestly  impossible  to  follow  out  the  pro- 
cess of  spore  formation  in  its  minutest  detail.  This  much, 
however,  can  as  a  rule  be  observed.  The  contents  of  the 
cell  are  at  first  homogeneous  and  the  first  indication  of  the 
beginning  of  sporulation  is  the  appearance  of  very  fine 
granules  in  the  protoplasm.  These  are  sometimes  spoken 
of  as  sporogenic  granules.  Some  of  these  are  larger  than 
others.  One  of  these  located  at  a  certain  place  in  the  cell 
gradually  increases  in  size  probably  because  the  other 
granules  gather  or  flow  together  at  this  point.  The  result 
is  a  roundish  or  ellipsoidal,  bright  body  which  at  first  has  no 
definite  envelope  or  wall.  Presently,  however,  a  distinct 
spore-wall  does  form  which  may  be  due  to  a  condensation 
of  the  protoplasm  of  the  cell  around  the  central  body.  At 
all  events  the  protoplasm  of  the  cell  disappears,  as  can  be 
shown  by  plasmolysis,  and  in  part  at  least  makes  up  the 
substance  of  the  spore.  Occasionally  a  very  small  resi- 


THE  LIFE  HISTORY  OF   BACTERIA.  49 

due  of  the  protoplasm  may  remain  on  the  outside  of   the 
spore. 

The  spore,  therefore,  may  be  considered  as  the  con- 
densed cell  contents.  It  contains  all  the  proteins  of  the 
parent  cell,  and,  when  completed,  lies  surrounded  by  an 
aqueous  liquid  inside  the  otherwise  empty  shell  or  cell- 
membrane.  This  original  cell  wall  soon  softens  and  dis- 
solves and  the  spore  is  thus  set  free.  Free  spores  are 
frequently  met  with  in  about  24  hours,  when  the  growth 
occurs  under  the  most  favorable  conditions.  Usually,  how- 
ever, they  are  not  liberated  for  several  days.  It  not  infre- 
quently happens  that  all  the  cells,  composing  a  thread,  form 
spores  at  the  same  time.  In  that  case  the  bright,  highly 
refracting  spores  are  present,  one  in  each  cell,  and  may  be 
compared  to  a  string  of  beads.  With  the  exception  of  two 
or  three  doubtful  cases,  it  may  be  stated  as  a  general  rule 
that  a  bacillus  gives  rise  to  but  one  spore.  Inasmuch  as 
the  protoplasm  of  the  parent  cell  goes  to  make  up  the 
spore  it  follows  that  the  former  ceases  to  exist  as  soon  as 
the  spore  is  fully  developed. 


c 


FIG.  12.    Sporulation.    a— First  stage  showing  sporogenic  gran- 
ules;   b — Incomplete  spore;    c — Fully  developed  spore. 

In  a  given  species  the  spore  nearly  always  develops  in 
the  same  relative  position  within  the  cell.  Thus,  in  some 
species  the  spore  forms  in  the  middle  and  occupies,  there- 
fore, a  median  position.  In  others  it  develops  at  the  very 
end  and  this  position  may  be  designated  as  terminal.  Again, 
the  spore  may  be  located  between  these  positions,  in  which 
case  it  is  said  to  be  intermediate  (Fig.  13  a,  6,  c). 

The  form  of  the  parent  cell  is  very  often  unchanged  by 
the  development  of  the  spore  on  the  inside.  At  other  times 
a  slight  enlargement  may  result  in  that  portion  of  the  cell 
occupied  by  the  spore.  Sometimes  this,  enlargement  is  very 


50  BACTERIOLOGY. 

marked  and  characteristic  forms  result.  Thus,  in  the 
case  of  a  median  spore  a  marked  enlargement  of  the  central 
portion  of  the  cell  gives  rise  to  a  spindle-shaped  form 
which  is  known  as  a  clostridium  (Fig.  13  a  2).  When  the 
spore  is  terminal,  a  corresponding  spherical  enlargement  of 
the  end  gives  rise  to  what  is  known  as  the  "drum-stick" 
bacillus  (Fig.  13  c  2). 


FIG.  13.  Position  of  spores;  resultant  forms  (diagrammatic). 
a— Median  spores;  £— Intermediate  spores;  c— Terminal  spores; 
2  a,  b,  c — Change  in  form  of  cell  due  to  the  presence  of  the  spore; 
2  a— Clostridium;  2  c — "  Drum-stick  "  form. 

In  the  case  of  motile  bacteria  spore  production  is 
usually  accompanied  by  a  loss  of  motion.  This  is  especially 
true  of  the  aerobic  species.  On  the  other  hand  the  anaero- 
bic bacteria  may  continue  to  move  about  for  some  time 
after  the  spore  has  fully  developed.  Flagella  can  be 
demonstrated  on  these  motile  spore-bearing  rods.  The 
persistence  of  motion  in  such  cases  may  be  due  to  the 
remnant  of  protoplasm  which  is  left  outside  of  the  spore 
and  which  lines  the  cell  wall. 

Spore  formation  has  been  supposed  to  take  place  when- 
ever the  culture  medium  became  exhausted.  This  view, 
however,  can  be  easily  shown  to  be  incorrect.  The  nour- 
ishing material  of  the  soil  is  by  no  means  consumed  when 
a  given  organism  begins  to  form  spores.  There  may  be 
times,  however,  when  this  factor  may  come  into  play. 
Under  the  artificial  conditions  of  the  laboratory  it  is  more 
likely  that  the  accumulation  of  the  waste-products  of  the 
•organism  and  the  action  of  these  products  on  the  living 
cell  cause  it  to  pass  into  the  spore  form.  As  long  as  the 


THE  LIFE  HISTORY  OF   BACTERIA.  51 

organism  is  growing-  under  the  best  possible  conditions  it 
will  not  give  rise  to  spores.  If,  for  instance,  it  is  trans- 
planted every  few  hours  in  order  to  avoid  the  formation  of 
waste  products  it  can  be  maintained  indefinitely  in  the 
vegetating  condition.  It  is  for  this  reason  that  bacteria 
growing  in  the  blood  of  a  living  animal  do  not  form  spore 

The  temperature  is  an  important  factor  in  spore  pro- 
duction. The  formation  of  spores  has  not  been  observed 
to  occur  below  16°. !  The  optimum  temperature  is  at  30 — 35°. 
Certain  bacteria  will  not  form  spores  in  the  absence  of  oxy- 
gen, whereas  other  bacteria  require  very  little  or  no  oxygen. 

The  composition  of  the  soil  has  much  to  do  with  sporu- 
lation.  The  presence  of  calcium  salts  and  the  absence  of 
pepton  favor  spore  production.  On  the  other  hand,  the 
presence  of  minute  amounts  of  carbolic  acid,  or  of  mercuric 
chloride  will  so  alter  an  organism  as  to  give  rise  to  a  spore- 
less  variety.  Asporogenic  bacteria  may  also  result  from 
prolonged  artificial  cultivation  on  the  ordinary  laboratory 
media.  The  anthrax  bacillus  which  has  been  cultivated  for 
some  years  never  gives  rise  to  spores  when  grown  on  the  ordi- 
nary media.  The  failure  in  spore  production  in  such  instan- 
ces is  similar  to  the  loss  of  motion  of  certain  bacteria  under 
like  conditions  (p.  38).  The  asporogenic  varieties  may  be 
compared  to  the  highly  cultivated,  seedless  flowering  plants. 

In  its  earliest  stage,  the  spore  appears  within  the  cell 
as  a  bright,  oil-like,  refracting  body.  This  appearance 
becomes  well  marked  when  the  spore  is  fully  developed, 
that  is  to  say,  when  the  spore-mass  has  surrounded  itself 
with  its  characteristic  spore-wall.  It  is  especially  marked 
in  the  free  spore.  The  spore,  as  a  rule,  possesses  the  same 
width  as  the  parent  cell.  It  is  a  short  oval  or  roundish 
body  and  can,  as  a  rule,  be  readily  recognized  by  its  micro- 
scopical appearance  and  by  its  behavior  to  stains. 

Unless   otherwise   indicated  the   temperatures  given  in  this 
work  are  Centigrade. 


52  .  BACTERIOLOGY. 

Owing-  to  the  peculiarly  dense  character  of  the  spore- 
wall  the  anilin  dyes  do  not  readily  stain  the  spore.  On 
staining,  therefore,  it  appears  as  a  colorless  body  imbedded 
in  a  stained  cell,  or  rather  within  the  stained  cell-wall. 
The  -presence  of  a  bright,  refracting  body  within  an 
unstained  cell,  or  the  presence  of  a  colorless,  non- staining 
body  in  a  stained  cell  does  not  prove  that  it  is  a  spore. 
Such  spore-like  bodies,  which  may  be  termed  pseudo-spores, 
have  been  frequently  observed,  and  mistaken  for  spores. 
Thus,  the  so-called  spores  of  the  tubercle  bacillus  are  due 
to  differences  in  the  density  of  the  protoplasm  which  con- 
tracts into  beads  or  balls,  thus  leaving  vacuoles  or  clear 
spaces  filled  with  a  cell  fluid.  The  small  bright  polar 
bodies  which  are  frequently  seen  in  glanders,  diphtheria, 
typhoid  and  other  bacilli  are  to  be  considered  as  small 
masses  of  condensed  protoplasm  since  they  stain  more 
deeply  than  the  surrounding  contents.  The  resistance  to 
destruction  is  a  valuable  criterion  of  a  spore-like  body,  but 
even  this  does  not  prove  its  spore  character.  The  true 
character  of  a  spore  can  only  be  established  by  observing 
its  germination. 

Spore  germination. — The  spore  which  is  formed  in  a  given 
culture  medium  does  not  germinate  until  after  it  has  been 
transplanted  to  a  good  new  soil.  The  original  medium  is 
unfit  for  this  purpose  largely  because  of  the  presence  of 
various  chemical  products,  which  were  elaborated  by  the 
vegetating  or  growing  organisms.  Moreover,  it  was  the 
presence  of  these  products- which  stimulated  the  cells  to 
form  spores.  In  a  suitable  soil,  and  under  proper  condi- 
tions, the  spore  gives  rise  to  a  young,  growing  cell,  which 
rapidly  increases  in  size,  reaches  what  may  be  called  the 
adult  state,  and  multiplies.  Eventually,  this  progeny  of 
vegetating  cells  gives  rise  to  new  spores. 

Like  the  seed  of  higher  plants,  the  spore  itself  does  not 
multiply.  Under  no  condition  will  one  spore  divide  and  give 


THE  LIFE  HISTORY  OF    BACTERIA.  53 

rise  to  two  or  more  spores.  All  that  it  can  do  is  to  repro- 
duce the  type  from  which  it  originally  developed.  The 
germinating  spore  gives  rise  to  one  young-  cell,  and,  inasmuch 
as  a  fully  developed  cell  produces  only  one  spore  and  then 
dies,  it  follows  that  spore  production  or  germination  is  in 
no  wise  a  means  of  multiplication.  The  spore  is  therefore 
solely  a  reproductive  or  perpetuating  form. 

The  process  of  germination  of  spores  is  not  the  same  for 
all  species.  It  will  vary,  more  or  less,  with  different  types, 
but  there  is  reason  to  believe  that  it  is  always  constant  for 
a  given  species.  The  mode  of  germination,  consequently, 
might  be  made  use  of  in  classifying-  bacteria  but,  unfortu- 
nately, it  requires  a  very  careful  and  prolonged  observation. 
For  this  reason,  the  study  of  the  germination  of  spores  is 
very  rarely  resorted  to.  The  process  has  been  followed  out 
in  only  a  relatively  small  number  of  bacteria,  probably  in 
not  more  than  10  or  12  species. 

The  first  change  observed  when  the  spore  is  about  to 
germinate  is  that  it  swells  up  and  lengthens.  This  increase 
in  size  is  probably  due  to  imbibition  of  moisture.  As  the  spore 
enlarges  it  becomes  less  bright.  The  marked  refraction 
disappears  and  gives  place  to  a  dull  appearance.  The  altered 
spore  now  gives  rise  to  a  young  cell  in  one  of  three  ways. 

1.  Direct  germination. — The  spore  gradually  changes, 
by  elongating  and  growing,  directly  into  a  new  cell.  In 
this  case  no  shell  or  spore-wall  is  thrown  off,  as  such,  but 
it  may  possibly  .be  softened  and  dissolved  during  the  pro- 
cess of  conversion.  Bearing  in  mind  the  development  of 
the  spore,  as  a  condensation  of  the  cell  protoplasm,  it 
would  seem  that  this  method  of  germination  was  the 
reverse  of  sporulation.  It  has,  however,  been  observed 
in  but  one  or  two  cases  (Fig.  14  a). 

Usually  the  spore-wall  splits  open  at  some  one  place 
on  the  surface  and  through  the  opening  thus  produced  the 
young  cell  makes  its  appearance.  The  opening  in  the 
spore-wall  may  be  at  one  or  both  ends,  that  is  to  say  polar, 


54  BACTERIOLOGY. 

or  it  may  be  across  the  middle,  in  which  case  it  is  desig- 
nated as  equatorial. 

2.  Polar  germination.  —  The   elongated   spore  opens  at 
one  pole  and  the  young  cell  thus  passes  out.     In  this  case 
the  long  axis  of  the  young  cell  is  parallel  to  the  long  axis 
of  the  spore.     The  anthrax  spore  germinates  in  this  way 
(Fig.  14  &). 

3.  Equatorial  germination.  —  The  lengthened  spore  opens 
as  the  result  of  a  split  across  the  middle.     In  some  forms, 
as  the  B.  megaterium,  the  spore-wall  is  divided  into  two, 
and  the    two   halves   are    pushed    asunder    by  the   young 
cell.     In  the  case  of  the  B.  subtilis  the  cleft  is  incomplete. 
In  such  instances  the  young  rod  leaves  the  spore-wall  either 
by  doubling  up,  forming  a  horse  shoe  as  it  were,  or  it  rotates 
and  passes  out  at  right  angles  to  the  long  axis  of  the  spore 
(Fig.  14  c,  d,  e). 


o  0  C  CH  0™ 


0   0   fi 


fll 


n 
u 


FIG.  14.  Spore  germination,  a.— Direct  conversion  ef  a  spore  into  a  bacillus  without 
the  shedding  of  a  spore-wall  (B.  leptosporus);  b — Polar  germination  of  B.  anthracis;  c. — 
Equatorial  germination  of  B.  subtilis;  d.— Same  of  B.  megaterium;  e.— Same  with  "  horse- 
shoe "  presentation. 

In  addition  to  a  suitable  soil,  the  germination  of  a  spore 
is  markedly  influenced  by  the  temperature.  The  process 
will  take  place  slowly  at  a  low  temperature,  and  more  rap- 
idly at  or  near  the  temperature  of  the  body.  In  the  latter 
case  it  may  require  from  3  to  5  hours.  When  a  mass  of 
spores  is  present,  the  germination  of  some  may  be  greatly 
delayed  as  compared  with  others.  This  fact  must  be  taken 
into  account  in  the  sterilization  of  culture  media. 

Structure  of  the  spore. — A  rather  dense,  impenetrable 
envelope  or  membrane  invests  the  contents  of  the  spore. 


THE  LIFE   HISTORY  OF   BACTERIA.  55 

The  prime  object  of  this  spore-wall  is  to  protect  the  proto- 
plasmic contents  against  conditions  which  would  otherwise 
destroy  the  life  of  the  organism.  The  exact  chemical  com- 
position of  the  spore-wall  is  not  known,  but  it  is  assumed 
to  be  protein  in  nature.  During-  germination  it  usually  soft- 
ens or  gelatinizes,  and  in  some  cases  becomes  thicker  at  the 
poles  (Fig.  14  c). 

The  contents  of  the  spore,  as  seen  from  the  method  of 
formation,  are  essentially  those  of  the  parent  cell,  less 
water.  Consequently  the  spore  substance,  which  may  be 
considered  as  condensed  protoplasm,  contains  all  the  pn> 
teins  of  the  original  cell.  Inasmuch  as  the  bacterial  cell 
contains  more  or  less  fat  it  follows  that  the  spore  will  like- 
wise possess  fatty  compounds.  The  bright,  highly  refrac- 
tive appearance  of  the  spore  is  highly  suggestive  of  the 
presence  of  an  oil-like  body.  When  spores  are  treated  with 
ether  no  fat  can  be  extracted,  but  this  does  not  justify  the 
assumption  that  fat  is  absent.  When  ether  is  shaken  up 
with  milk  it  will  not  remove  the  fat  which  is  present  in  the 
milk  globules. 

Spores  are,  as  a  rule,  perfectly  colorless.  In  a  few 
fluorescing  bacteria  red  spores  have  been  observed.  In 
several  other  instances  markedly  green  spores  have  been 
met  with. 

The  anilin  dyes  do  not  stain  spores  as  readily  as  the 
vegetative  form.  This  is  commonly  believed  to  be  due  to  the 
dense  spore-wall,  which  hinders  the  penetration  of  the  dye. 
Undoubtedly,  the  contents  of  the  spore  will  take  up  the 
stain  very  slowly  owing  to  the  almost  total  absence  of 
water.  For  the  same  reason,  the  contents,  when  once  stained, 
can  be  decolored  only  with  difficulty.  The  method  of 
double  staining  spores  will  be  described  in  Chapter  X. 

Spores  are  characterized  by  their  extreme  resistance  to 
destruction.  The  vegetating  form  is  readily  killed  by  mere 
desiccation,  whereas  the  spore  can  be  kept  in  the  dry  con- 
dition for  years.  Again,  the  vegetating  form  is  killed  in  a 


56  BACTERIOLOGY. 

few  minutes  by  exposure  to  moist  heat  of  about  70°,  but  the 
spore  form  may  resist  for  hours.  Steam  heat,  which,  as  a 
rule,  instantly  kills  the  vegetating  form  will  require  5  to  10 
minutes,  and  in  some  instances  as  many  hours,  to  destroy 
the  spore.  A  similar  difference  between  the  vegetating 
and  spore  form  will  be  observed  in  the  action  of  various 
chemical  substances. 

Spores  will  resist  the  action  of  dry  heat  more  readily 
than  that  of  steam  heat.  A  dry  heat  of  140°  may  require 
an  hour  or  more  to  destroy  spores  which  would  be  killed  in 
a  few  minutes  by  exposure  to  steam.  Heat  and  moisture  are 
therefore  more  -destructive  than  heat  alone.  This  is  equally 
true  for  the  higher  forms  of  life.  Thus,  in  a  very  dry  cli- 
mate man  can  endure  without  discomfort  a  temperature  of 
130-140°  F.  whereas  a  much  lower  temperature  in  the  pres- 
ence of  moisture  is  prostrating. 

Steam  heat  under  pressure  will  destroy  spores  more 
readily  than  will  ordinary  steam  heat.  The  spores  of  a 
certain  potato  bacillus,  which  resist  steam  for  5-6  hours, 
are  destroyed  in  10  minutes  by  an  exposure  to  steam  under 
pressure  at  a  temperature  of  120°. 

The  spores  of  different  species  possess  different  degrees 
of  resistance.  Thus,  the  anthrax  spores  are  more  easily 
destroyed  than  those  of  the  hay  bacillus.  Again,  the  seve- 
ral varieties  of  a  given  species  may  produce  spores  which 
will  show  extreme  variation  in  resistance.  There  are  an- 
thrax spores  which  are  readily  destroyed  by  5  per  cent,  car- 
bolic acid  within  24  hours,  whereas  other  anthrax  spores 
may  not  be  affected  by  an  exposure  of  40  or  50  days  to  this 
solution. 

The  extreme  resistance  of  some  spores,  as  briefly  indi- 
cated above,  accounts  for  the  theory  of  spontaneous 
generation  which  at  one  time  was  quite  universally  accept- 
ed. According  to  this  theory  it  was  supposed  that  the 
lower  forms  of  animal  and  plant  life  could  develop  without 


THE   LIFE   HISTORY  OF  BACTERIA.  57 

the  agency  of  other  organisms;  in  other  words,  that  life 
could  originate  directly  from  dead  matter.  This  view  was 
apparently  substantiated  by  the  fact  that  when  an  infusion 
of  wheat  or  barley  was  boiled  in  a  closed  flask  for  some 
minutes  and  then  set  aside  various  bacteria  would  develop 
in  the  liquid.  The  exposure  to  the  temperature  of  boiling 
wTater  was  assumed  to  be  sufficient  to  destroy  all  living 
matter  and  consequently  any  new  life  that  might  subse- 
quently develop  must  come  from  dead  matter.  Such  was 
the  theory  of  spontaneous  generation  which,  formulated 
by  Needham  in  1747,  persisted  as  a  dominant  idea  for  more 
than  100  years. 

This  theory  existed  because,  in  the  first  place,  it  was 
not  known  that  bacteria  were  to  be  found  almost  everywhere 
in  nature.  In  the  second  place,  the  existence  of  spores  and 
the  remarkable  degree  of  resistance  which  they  possessed 
was  not  dreamed  of.  The  researches  of  Pasteur  (1861)  first 
demonstrated  the  wide  distribution  of  bacteria  in  the  air  and 
elsewhere,  and,  above  all,  clearly  proved  the  existence  of 
highly  resistant  forms  of  bacteria,  the  spores.  A  moderate 
amount  of  boiling  will  kill  all  vegetating  forms  and  some 
feeble  spores.  The  highly  resistant  spores  are  not  destroyed 
and  hence  subsequently  germinate.  The  beginner  in  the  lab- 
oratory will  invariably  meet  with  instances  of  so-called 
spontaneous  generation  whenever  the  culture  media  have 
not  been  properly  sterilized.  The  smallest  of  living  beings, 
like  the  highest  forms  of  life,  are  always  descended  from 
antecedents  of  their  own  kind.  While  it  may  not  be  true 
to  say  that  all  life  comes  from  the  egg,  it  is  true  that  "  all 
life  comes  from  life." 


CHAPTER    IV. 
THE  ENVIRONMENT  OF  BACTERIA. 

Although  bacteria  are  present  almost  everywhere  upon 
the  surface  of  the  globe  it  must  not  be  supposed  that  they 
are  capable  of  growing  wherever  they  may  be  found.  The 
air  always  contains  a  greater  or  less  number  of  bacteria, 
but  these  organisms  are  not  multiplying.  They  are  present 
merely  as  extremely  minute  particles  picked  up,  and  wafted 
about  by  currents.  The  conditions  met  with  in  the  air  are 
far  from  being  favorable  to  their  development.  On  the 
contrary  the  desiccation  or  drying,  to  which  the  floating 
bacteria  are  subjected,  is  directly  injurious  to  these 
organisms.  The  sporeless  bacteria  are  probably  speedily 
destroyed  as  a  result  of  desiccation  and  of  the  action  of 
sun-light. 

Inasmuch  as  bacteria  are  specifically  heavier  than  air 
they  tend  to  settle  together  with  the  coarser,  floating  matter. 
The  dust  which  therefore  deposits  on  the  floor,  windows, 
or  upon  the  clothes,  hair,  or  skin  is  rich  in  various  bacteria, 
yeasts  and  moulds.  But  even  when  thus  deposited  these 
organisms  will  not  grow  and  multiply.  In  order  that  they 
shall  grow  it  is  necessary  that  they  reach  a  suitable  nutri- 
ent medium  and  an  essential  condition  of  a  nutrient  soil  is 
that  it  shall  contain  moisture. 

Moisture,  consequently,  is  absolutely  necessary  for  the 
development  of  bacteria.  More  than  that,  it  is  necessary  to 
the  well  being  of  every  form  of  life,  whether  high  or  low. 
This  is  seen  in  the  fact  that  water  enters  largely  into 
the  composition  of  living,  growing  protoplasm.  The  tis- 
sues of  higher  plants  and  animals  contain  from  70  to  80 


THE  ENVIRONMENT   OF   BACTERIA.  59 

per  cent,  of  water.  The  organisms  which  grow  in  water, 
like  algae  and  fish,  contain  a  relatively  higher  per  cent. 
As  indicated  above  bacteria  live  only  in  liquids  and  on 
moist  surfaces.  They  are,  therefore,  essentially  aquatic 
and,  as  such,  they  hold  within  their  cells  a  large  amount  of 
water. 

The  chemical  examination  of  various  actively  growing 
bacteria  shows  that  they  contain  about  85  per  cent,  of 
water.  The  proteins,  which  are  necessary  constituents  of 
protoplasm,  make  up  about  10  per  cent.  The  amount  of 
fatty  substances  present  will  vary  with  different  species. 
On  an  average  they  may  be  said  to  contain  about  one  per 
cent,  of  fat.  Certain  bacteria,  like  those  of  glanders  and 
tuberculosis,  are  very  rich  in  fats,  which  may  constitute  as 
much  as  40  per  cent,  of  the  dried  cells.  The  carbohydrate 
group,  represented  by  cellulose,  granulose  or  dextrin-like 
compounds,  is  at  times  present.  The  inorganic  or  mineral 
constituents  make  up  about  one  per  cent,  of  the  living 
organism. 

In  addition  to  the  above  components  of  the  bacterial 
cell  other  substances  may  at  times  be  present.  The  disease- 
producing  bacteria  elaborate  within  their  cells  the  specific 
poison  or  toxin.  All  bacteria,  moreover,  contain  one  or 
more  soluble  ferments  or  enzymes. 

Carbon. — The  contents  of  the  living  cell  are  made  out 
of  the  food  on  which  the  organism  lives.  The  majority  of 
bacteria  differ  from  the  higher  plants  in  one  marked 
respect,  and  that  is,  that  they  do  not  contain  chlorophyll. 
It  is  by  the  aid  of  chlorophyll,  in  the  presence  of  sun-light, 
that  the  higher  plant  assimilates  the  carbonic  acid  of  the 
air.  The  carbon  is  retained  by  the  plant  while  the  oxygen 
is,  as  it  were,  exhaled  and  returned  to  the  atmosphere. 
Consequently,  the  source  of  the  carbon,  present  in  the 
higher  plant  as  protein  matter,  cellulose,  starch,  fats, 
etc.,  is  the  simple,  inorganic  compound,  the  carbonic  acid 


60  BACTERIOLOGY. 

of  the  air.  Owing-  to  the  absence  of  chlorophyll  the 
majority  of  bacteria  cannot  utilize  this  source  of  carbon 
They  are,  as  a  rule,  dependent  upon  organic  compounds. 
That  is  to  say,  the  more  or  less  complex  carbon  compounds 
elaborated  by  higher  plant  and  animal  life  constitute  the 
food  out  of  which  the  bacteria  can  appropriate  the  carbon 
and  build  up  their  own  protoplasm.  To  a  very  large  ex- 
tent, therefore,  bacteria  depend  for  their  food  upon  dead 
animal  and  vegetable  matter.  In  this  respect  they  resemble 
animal  life.  As  is  well  known  the  animal  cannot  utilize 
the  carbonic  acid  of  the  air  but  obtains  its  carbon  from  the 
preformed  carbon  compounds  in  the  animal  or  vegetable 
food.  The  proteins,  carbohydrates  and  fats  supply  the 
necessary  carbon  to  the  growing  animal  and  also  to  the 
vegetating  bacteria.  The  latter  may  also  obtain  their 
carbon  from  more  simple  organic  compounds  such  as 
glycerin,  or  lactic,  or  tartaric  acids.  It  may  be  interesting 
to  note  that  bacteria,  like  animals,  instead  of  assimilating 
actually  give  off  carbonic  acid. 

The  above  is  true,  undoubtedly,  for  the  majority  of  bac- 
teria. There  are,  however,  certain  bacteria  which  can  live 
on  wholly  inorganic  matter.  The  interesting  group  of  nitro- 
bacteria,  although  they  do  not  contain  chlorophyll,  are  nev- 
ertheless able  to  assimilate  carbonic  acid  even  in  the  absence 
of  sun-light.  Organisms  of  this  type  may  well  be  consid- 
ered as  belonging1  to  the  earliest  inhabitants  of  the  globe. 

Nitrogen. -^Carbon  is  a  characteristic  and  essential  con- 
stituent of  protoplasm.  There  are  other  elements  equally 
important,  and  among  these  nitrogen  deserves  especial  at- 
tention. Although  the  air  contains  nearly  80  per  cent,  of 
free  nitrogen,  the  higher  plant  cannot  obtain  this  element 
from  this  source.  All  the  nitrogen  which  enters  into  the 
composition  of  the  higher  plants,  with  certain  exceptions 
presently  to  be  mentioned,  is  derived  from  various  nitrogen 
compounds  present  in  the  soil.  The  ammonia,  nitrous  and 


THE   ENVIRONMENT   OF   BACTERIA.  61 

nitric  acids  present  in  the  earth  are  absolutely  essential  to 
the  growth  and  development  of  most  of  the  higher  plants. 
This  form  of  life,  therefore,  obtains  all  of  its  nitrogen  from 
inorganic  compounds.  The  animal  organism,  on  the  other 
hand,  cannot  utilize  these  compounds  as  food.  The  nitro- 
gen which  is  necessary  to  the  building  up  of  protoplasm  in 
the  animal  cell  is  derived  from  preformed,  organic,  nitrogen, 
containing  substances.  Moreover,  only  certain  nitrogen- 
ous, organic  compounds  can  serve  as  food.  These  are  the 
protein  or  albuminous  substances  which  have  been  made  by 
the  living  plant  or  animal  cell.  Animal  life  is  therefore 
dependent  upon  plant  life  for  its  supply  of  nitrogen. 

Bacteria,  like  animal  organisms,  may  obtain  the  nitrogen 
necessary  for  their  growth  from  organic,  nitrogenous  sub- 
stances. The  proteins  present  in  dead  animal  or  vegetable 
matter  constitute,  therefore,  an  important  source  of  nitrogen 
for  these  lower  forms  of  plant  life.  It  must  not  be  inferred, 
however,  that  the  proteins  are  the  only  source,  for  such  is 
by  no  means  the  case.  Many  bacteria  can  obtain  their  nec- 
essary nitrogen  from  compounds  that  are  vastly  more  .sim- 
ple than  the  proteins.  Thus,  the  amido  acids,  such  as 
asparagin,  constitute  an  excellent  food  in  this  respect  for 
certain  bacteria.  Furthermore,  many  bacteria  may  obtain 
their  nitrogen  from  wholly  inorganic  compounds,  such  as 
ammonium  chloride.  There  are  other  species  which  can 
thrive  better  on  nitrates  than  on  other  forms  of  nitrogen. 

The  nodules  or  tubercles  which  are  found  upon  the  roots 
of  leguminous  plants,  such  as  the  pea,  lupine,  etc.,  are  essen- 
tially masses  of  certain  bacteria,  or  bacteroids,  which  possess 
the  remarkable  property  of  assimilating  the  free  nitrogen 
of  the  air  and  in  transmitting  this  element  to  the  growing 
plant.  A  vigorous,  healthy  growth  of  such  plants  is  directly 
dependent  upon  the  presence  of  these  parasitic  bacteria. 

It  is  -evident,  therefore,  that  bacteria  can  obtain  their 
nitrogen  from  various  sources.  This  element  may  be  appro- 
priated from  the  complex  organic  protein  molecule,  or  from 


62  BACTERIOLOGY. 

the  simpler  amido  acids,  or  from  strictly  inorganic  com- 
pounds, such  as  ammonium  salts,  nitrates  or,  in  rare  cases, 
free  nitrogen.  The  fact  that  some  species  can  thrive  better 
than  others  upon  one  of  these  sources  of  nitrogen  can  be 
made  use  of  in  differentiating  one  form  from  another.  Thusj 
the  typhoid  bacillus  cannot  utilize  the  nitrogen  in  ammonium 
salts,  whereas  the  colon  bacillus  can.  Artificial  culture 
media  (see  Uschinsky's  medium)  can  be  prepared  which  con- 
tain chiefly  inorganic  constituents  and  only  simple  organic 
compounds,  such  as  lactic,  or  tartartic  acids,  or  glycerin,  or 
glucose.  The  latter  compound  is  especially  useful  as  a 
source  of  carbon  under  these  conditions  of  growth. 

The  hydrogen  which  enters  into  the  composition  of  pro 
toplasmis  probably  derived  along  with  the  carbon  from  the 
organic  food  constituents.  Whether  it  can  be  obtained  from 
inorganic  compounds,  such  as  ammonia  and  water  is  not 
known.  Water,  as  such,  is  taken  up  by  the  cell  from  the 
surrounding  medium  and,  moreover,  may  be  formed,  in  part, 
like  carbonic  acid,  by  oxidation  changes  within  the  organ- 
ism. 

In  addition  to  carbon,  nitrogen  and  hydrogen  every  liv- 
ing organism  must  be  supplied  with  oxygen.  The  higher 
animal  obtains  its  oxygen  from  the  air,  whereas  higher 
plants  are  obliged  to  depend  upon  the  oxygen  contained  in 
water,  nitrous  and  nitric  acids.  The  majority  of  bacteria 
can  probably  utilize  the  oxygen  of  the  air,  which  is  seen  in 
the  fact  that  they  grow  only  in  the  presence  of  air.  Other 
bacteria,  as  will  presently  be  seen,  thrive  only  in  the  ab- 
sence of  air.  In  such  cases  the  necessary  oxygen  is  undoubt- 
edly obtained  from  organic  compounds  such  as  proteins,  or 
carbohydrates. 

Certain  inorganic  salts  or  mineral  constituents  are  like- 
wise necessary  to  the  well-being  of  living  organisms.  Bacte  - 
ria  may  obtain  their  sulphur  and  phosphorus  from  preformed 
organic  compounds  such  as  the  proteins.  They  can,  however, 


THE  ENVIRONMENT  OF  BACAERIA.  63 

appropriate  these  elements  from  inorganic  sulphates  and 
phosphates.  The  various  chlorides  found  in  nature  furnish 
the  necessary  chlorine,  and  incidentally  such  metals  like 
sodium  and  potassium.  The  traces  of  calcium,  iron,  and 
other  metals  present  in  various  liquids  are  sufficient,  as  a 
rule,  to  meet  the  requirements  of  an  organism. 

The  amount  of  mineral  constituents  necessary  to  sup- 
port bacterial  life  is  exceedingly  small.  As  shown  above, 
the  ash  constitutes  only  about  one  per  cent,  of  the  growing 
bacteria.  It  has  been  estimated  that  1  mg.  of  living  bacteria 
contains  about  30,000,000,000  cells,  in  which  case  this  large 
number  of  organism  will  yield  only  rta  mg.  of  ash.  Inasmuch 
as  the  organic  constituents  of  bacteria  make  up  less  than 
15  per  cent,  of  the  whole  it  will  be  seen  that  the  amount  of 
organic  matter  necessary  as  food  is  likewise  exceedingly 
small.  This  is  shown  by  the  fact  that  many  bacteria  can 
multiply  not  only  in  ordinary  water,  but  even  in  distilled 
water. 

The  reaction  of  the  nutrient  medium  exerts  an  important 
influence  upon  the  growth  of  bacteria.  An  acid  reaction  is 
not  as  favorable  as  an  alkaline  one.  Thus,  an  acidity  cor- 
responding to  20  or  30  c.  c.  of  normal  acid  per  liter  will  inhibit 
the  growth  of  many  bacteria,  whereas  the  same  organisms 
will  thrive  in  a  medium,  the  alkalinity  of  which  corresponds 
to  50  c.  c.  of  normal  alkali  per  liter.  It  is  customary,  there- 
fore, to  cultivate  bacteria  upon  a  neutral  or  slightly  alka- 
line soil.  The  moulds,  on  the  other  hand,  seem  to  thrive 
best  upon  an  acid  medium. 

Organic  matter  derived  from  dead  animals  or  plants  can 
be  met  with  almost  everywhere.  The  simple  food  require- 
ments of  bacteria  are,  therefore,  widely  distributed  in  nature 
and  for  that  reason  these  organisms  will  be  found  almost 
universally  present  upon  the  surface  of  the  globe.  There 
are  but  few  places  where  bacteria  are  absent.  The  air  at  high 


64  BACTERIOLOGY. 

altitudes  and  that  in  mid-ocean  ;  the  air  exhaled  from  the  lungs; 
the  deeper  layers  of  the  soil  and  the  water  coming  from  such 
depths  ;  the  internal  fluids,  tissues  and  secretions  of  healthy,  nor- 
mal animals  and  plants  are  practically  the  only  places  in  nature 
where  bacteria  are  not  present. 

As  pointed  out  on  p.  58,  the  ordinary  air  always  contains 
bacteria  which,  as  minute  dry  particles,  are  carried  about  by 
the  'movements  of  the  atmosphere.  In  a  perfectly  tight 
room,  free  from  currents,  the  suspended  organisms  will 
readily  settle  to  the  floor,  owing  to  the  fact  that  they  are 
specifically  heavier  than  air.  A  similar  tendency  exists  in 
the  open  air  and  as  a  result  the  lower  layers  of  the  atmos- 
phere will  contain  more  bacteria  than  the  higher  ones. 
Moreover,  the  air  is  purified  by  the  falling  rain  or  snow, 
which  drag  down  to  the  earth  the  suspended  solid  particles. 
It  is  evident,  therefore,  that  the  atmosphere  on  the  tops  of 
high  mountains  will  be  practically  free  from  bacteria. 

The  air  in  mid-ocean  may  also  be  considered  as  free 
from  suspended  particles,  including  bacteria.  This  is  due  to 
the  washing,  as  it  were,  of  the  winds  or  currents  of  air  in 
their  passage  over  the  water.  The  bacteria  in  the  air  on 
coming  into  contact  with  a  moist  surface  are  held  back. 
This  fact  holds  true  not  only  in  the  case  of  the  air  which 
passes  over  large  surfaces  of  water  but  also  in  the  case  of 
expired  air.  No  matter  how  many  hundreds  or  thousands 
of  bacteria  may  be  present  in  the  air  which  is  drawn  into 
the  lungs  the  expired  air  is  always  practically  free  from 
organisms.  The  latter  adhere  to  the  moist,  mucous  mem- 
brane of  the  mouth,  nose,  throat  and  bronchi,  and  conse- 
quently, the  expired  air  contains  very  few  or  no  bacteria. 

It  may  be  stated  as  a  general  and  most  important  rule 
that  the  bacteria  present  in  liquids  or  on  moist  surfaces 
cannot  leave  such  places  and  enter  the  air.  The  most  deadly 
organism  can  be  studied  in  the  laboratory,  as  it  grows  on 
moist  culture  media,  without  any  danger  in  this  respect.  For 
the  same  reason,  the  breath  of  a  consumptive  is  free  from 


THE  ENVIRONMENT  OF   BACTERIA.  65 

the  dreaded  tubercle  bacillus  which  can  only  leave  the 
mouth,  with  the  sputum,  or  in  the  small  particles  of  liquid 
which  may  be  scattered  about  in  a  fit  of  violent  coughing-. 

The  surface  layers  of  the  soil  are  exceedingly  rich  in 
bacteria.  These  organisms  decrease  rapidly  in  numbers 
with  the  depth,  and  eventually  disappear  entirely.  The 
soil  at,  or  below,  a  depth  of  8  or  10  feet  can  be  regarded  as 
practically  free  from  bacteria.  This  is  due,  in  the  first 
place,  to  the  fact  that  the  earth  acts  as  a  filter  retaining 
most  of  the  organisms  at,  or  near,  the  surface.  It  is  also  due, 
in  part,  to  the  unfavorable  conditions,  such  as  low  tem- 
perature and  insufficient  supply  of  oxygen.  The  rain,  or 
melting  snow,  percolates  through  the  soil  and  eventually  be- 
comes wholly  free  from  the  suspended  organisms.  It  is  clear, 
therefore,  that  the  water  which  comes  from  the  deeper 
layers  of  the  earth  is  free  from  bacteria.  The  water  of  a 
spring,  or  of  a  tubular,  or  artesian  well  may  contain  some 
organisms,  but  these  are  due  to  accidental  contamination  at, 
or  near,  the  surface. 

The  higher  animal  inhales  every  day  an  enormous  num- 
ber of  bacteria.  Moreover,  these  organisms  are  constantly 
being  introduced  into  the  alimentary  canal  with  the  food. 
The  intestinal  contents,  as  a  result,  are  extremely  rich  in 
bacteria.  The  surface  of  the  body  may  also  harbor  a  large 
number  of  these  organisms.  In  spite  of  the  fact  that  the  body 
is  thus  besieged  on  all  sides  by  countless  bacteria,  it  is  never- 
theless free  from  them.  In  a  healthy  individual  the  blood 
-and  lymph,  the  various  internal  organs  and  tissues  are 
wholly  free  from  bacteria.  It  follows,  therefore,  that  the 
secretions,  such  as  the  urine  and  milk,  of  a  normal  individual 
are  perfectly  germ  free. 


66  BACTERIOLOGY. 

Saprophytic  and  Parasitic  Bacteria. 

Bacteria  are  grouped,  according-  to  their  habitat,  into 
saprophytic  and  parasitic.  The  saprophytic  bacteria  are 
those  which  live  on  dead  animal,  or  vegetable  matter.  The 
vast  majority  of  bacteria  belong-  to  this  group.  Only  a 
relatively  small  number  of  organisms  possess  the  power  of 
growing  in  the  living  animal  or  plant.  These  are,  there- 
fore, designated  as  parasitic  bacteria.  Because  the  latter 
grow  and  thrive  in  the  living  body  it  must  not  be  inferred 
that  they  subsist  entirely  on  living  matter.  The  waste 
products  of  the  living,  or  of  the  dead  cells  may  furnish  the 
necessary  food  supply.  The  parasitic  bacteria  include 
many  harmless  forms  in  addition  to  the  disease -producing 
organisms. 

The  majority  of  the  bacteria  present  in  the  mouth,  stom- 
ach and  intestines  cannot  be  regarded  as  parasitic.  They 
live  on  the  dead  matter  in  the  alimentary  tract  and  can  live 
equally  well  outside  of  the  body.  They  are  to  be  consid 
ered  as  saprophytic  bacteria.  A  few  of  the  mouth  bacteria, 
such  as  the  vibrios  and  spirals,  cannot  be  made  to  grow 
on  artificial  media.  They  are,  apparently,  dependant  upon 
the  living  organism.  This  is  equally  true  of  the  leprosy 
bacillus  and  to  a  certain  extent  of  the  germs  of  gonorrhea 
and  of  tuberculosis.  It  is  customary  to  designate  those 
bacteria  which  are  compelled  to  live  in  the  living  body  as 
obligative  parasites. 

On  the]  other  hand,  among  the  saprophytic  bacteria 
there  are  those  which  are  unable  to  thrive  in  the  living 
body.  They  are  compelled,  as  it  were,  to  live  on  the  dead 
matter  found  in  nature.  Consequently,  they  are  known  as 
obligative  saprophytes.  It  is  evident  that  the  obligative 
parasites  and  the  obligative  saprophytes  constitute  two 
extreme  groups.  Numerous  bacteria  occupy  an  intermedi- 


THE  ENVIRONMENT  OF  BACTERIA.  67 

ate  position  between  these  extremes.  There  are  bacteria 
which  may  primarily  be  considered  as  saprophytic,  but 
which,  at  times,  may  lead  a  temporary,  parasitic  existence. 
These  are  designated  as  facultative  parasitic  bacteria. 
Again,  certain  bacteria  which  are  primarily  parasitic  may 
be  able  to  thrive  somewhat,  outside  of  the  body,  on  dead 
matter.  They  elect,  so  to  speak,  a  saprophytic  life  and 
hence  are  known  as  facultative  saprophytes. 

The  transition  from  one  extreme  to  the  other  can  per- 
haps be  seen  best  from  the  following'  arrangement  of  the 
several  groups: 

Obligative  saprophytic  bacteria, 
Facultative  parasitic  bacteria, 
Facultative  saprophytic  bacteria, 
Obligative  parasitic  bacteria. 

The  parasitic  as  well  as  saprophytic  bacteria  are 
unable  to  utilize  carbonic  acid  as  a  food.  They  cannot 
build  up  protoplasm  out  of  wholly  inorganic  substances. 
In  other  words,  they  are  dependent  upon  organic  compounds 
which  were  made  by  animal  or  plant  life.  The  group  of 
nitro-bacteria  (p.  61)  constitutes  an  exception  to  this  rule, 
inasmuch  as  these  can  exist  on  wholly  inorganic  matter. 
Consequently  they  do  .not,  strictly  speaking,  belong  in  the 
group  of  saprophytic  bacteria. 

The  parasitic  bacteria,  as  mentioned  above,  include  the 
various  disease-producing  organisms.  The  saprophytic 
bacteria  include  those  forms  which  thrive  on  dead  matter, 
and  consequently  the  bacteria  which  produce  fermentation 
and  putrefaction  fall  under  this  head. 


68  BACTERIOLOGY. 

Oxygen  Requirements. 

It  was  at  one  time  generally  accepted  that  all  living- 
being's  required  free  oxygen.  In  1861,  Pasteur  described  as 
the  cause  of  butyric  acid  fermentation  a  bacillus  which 
could  be  cultivated  only  in  the  absence  of  oxygen.  He 
designated  those  organisms  which  required  oxygen  for  their 
development  as  aerobic;  whereas  those  which  lived  in  the 
absence  of  oxygen  were  designated  as  anaerobic.  The 
"  vibrion  butyrique  "  was  the  first  known  anaerobic  germ. 
In  1877,  Pasteur  demonstrated  that  an  experimental  disease, 
"septicemie"  or,  as  it  is  more  commonly  known,  malignant 
edema,  was  due  to  an  anaerobic  bacillus.  Since  then  about 
fifty  anaerobic  bacteria  have  been  described. 

An  examination  of  the  various  anaerobic  bacteria  will 
show  that  some  are  absolutely  dependant  upon  the  total 
exclusion  of  oxygen.  The  presence  of  a  small  amount  of 
air  will  promptly  stop  the  growth  of  such  organisms. 
These  remarkable  forms  can  be  designated  as  obligative 
anaerobes  since  they  are  obliged  to  live  under  strictly 
anaerobic  conditions.  On  the  other  hand,  there  are  aerobic 
bacteria  which  live  only  in  the  presence  of  an  abundance 
of  oxygen.  As  soon  as  the  amount  of  air,  or  oxygen  is 
diminished  growth  stops.  They  are  obliged  to  live  in  the 
presence  of  air  and,  for  this  reason,  they  are  termed  obliga- 
tive aerobes. 

The  obligative  aerobes  and  the  obligative  anaerobes  con- 
stitute the  two  extremes  as  far  as  oxygen  requirements  are 
concerned.  Most  of  the  bacteria  show  an  adaptability  to 
one  or  the  other  of  these  conditions,  and  as  a  result  the 
two  obligative  groups  are  connected  by  a  series  of- species 
which  can,  more  or  less  readily,  accommodate  themselves 
to  the  presence  or  absence  of  oxygen.  A  given  organism 
which  thrives  best  in  the  presence  of  air  may  grow,  though 
perhaps  less  abundantly,  in  the  absence  of  oxygen.  Such 


THE  ENVIRONMENT  OF  BACTERIA.  69 

an  organism  is  designated  as  a  facultative  anaerobe.  On 
the  other  hand,  a  germ  which  may  grow  in  the  presence  of 
air,  but  which  grows  best  under  anaerobic  conditions,  is 
known  as  a  facultative  aerobe.  The  transition  from  one 
extreme  to  the  other  can  be  seen  from  the  following  arrange- 
ment of  the  groups: 

Obligative  aerobic  bacteria, 
Facultative  anaerobic  bacteria, 
Facultative  aerobic  bacteria, 
Obligative  anaerobic  bacteria. 

The  ability  of  an  organism  to  grow  in  the  tissues  and 
fluids  of  the  body  indicates  that  it  can  thrive  in  the  more 
or  less  complete  absence  of  free  oxygen.  Consequently, 
most  of  the  disease-producing  bacteria  are  facultative  ana- 
erobes. When  grown,  however,  under  artificial  conditions 
they  may  show  an  almost  Obligative  aerobic  character. 
This  is  especially  true  of  the  cholera  vibrio. 

Although  many  bacteria  can  live  in  the  absence  of  free 
oxygen  it  must  not  be  supposed  that  they  are  wholly  inde- 
pendent of  that  element.  As  stated  heretofore,  living  pro- 
toplasm always  contains  proteins  and  these  compounds 
contain  oxygen  as  one  essential  constituent.  Consequent- 
ly, anaerobic  bacteria  require  oxygen  in  some  shape  or 
other.  The  fact  that  they  obtain  oxygen  from  some  source 
is  seen  not  only  in  the  chemical  composition  of  the  cell, 
but  also  in  the  fact  that  anaerobes,  like  other  bacteria, 
exhale  carbonic  acid.  Apparently,  anaerobic  bacteria  ob- 
tain the  necessary  oxygen  from  various  organic  compounds. 
The  proteins  can  be  utilized  for  this  purpose.  The  carbo- 
hydrates, especially  glucose,  are  valuable  in  this  respect. 
It  is  customary,  therefore,  to  grow  anaerobic  bacteria  on 
media  to  which  glucose  has  been  added. 

Obligative  anaerobic  bacteria  are  commonly  met  with 
in  the  soil.  They  are  present,  to  some  extent,  in  the  intes- 
tinal contents.  Only  one  organism  of  this  group  has  been 


70  BACTERIOLOGY. 

found  in  the  mouth,  in  a  decaying-  tooth  cavity.  It  would 
appear  to  be  somewhat  contradictory  to  find  anaerobic 
bacteria  growing-  in  the  soil  apparently  in  the  presence  of 
air.  Nevertheless,  such  is  the  case.  The  explanation  of 
this  curious  fact  is  very  simple  and  can  be  very  easily 
demonstrated  in  the  laboratory.  If,  for  example,  an  obli- 
gative anaerobic  germ  is  planted  in  beef  tea,  in  the  presence 
of  air,  it  will  not  develop.  If,  however,  an  aerobic  germ  is 
planted  at  the  same  time  as  the  anaerobe  both  organisms 
will  develop.  It  would  seem  as  if  the  aerobic  germ  con- 
sumes the  oxygen  in  the  immediate  neighborhood  of  the 
anaerobe  and  thus  enables  the  latter  to  grow.  This  favor 
ing  action  on  the  part  of  certain  bacteria  is  known  as 
microbic  association,  and  it  is  undoubtedly  because  of  such 
assistance  that  obligative  anaerobic  bacteria  are  able  to 
multiply  in  the  soil.  Moreover,  it  is  an  association  of  this 
kind  which  enables  certain  pathogenic  members  of  this 
group,  such  as  the  tetanus  bacillus,  to  develop  in  the  body 
of  animals. 

With  but  one  or  two  exceptions,  all  the  known  obliga- 
tive anaerobic  bacteria  are  bacilli.  An  obligative  anaerobic 
micrococcus  has  been  isolated  from  pus.  This  group  is 
characterized  by  the  production  of  large  quantities  of  gas. 
Moreover,  butyric  acid  is  a  very  common  product  and  may 
be  detected  by  the  odor,  not  only  in  the  artificial  cultures, 
but  at  times  even  in  the  body  of  an  animal  infected  with 
certain  organisms  of  this  group.  The  obligative  anaerobic 
bacteria,  therefore,  exhibit  a  marked  ability  to  produce 
fermentation. 

The  methods  of  cultivating  anaerobic  bacteria  will  be 
described  in  connection  with  these  organisms,  (Chapter  XI). 


THE  ENVIRONMENT  OF   BACTERIA.  71 

Temperature. 

In  addition  to  a  suitable  nutrient  soil  and  a  proper 
supply  of  oxyg-en,  bacteria  require  a  certain  temperature 
for  their  development.  As  with  other  forms  of  life,  each 
bacterial  species  has  a  minimum,  optimum  and  maximum 
temperature  at  which  it  will  develop.  In  some  species  the 
range  of  temperature  at  which  they  can  grow  is  very 
limited.  Thus,  in  the  case  of  the  tubercle  bacillus  and  the 
gonococcus  the  maximum  and  minimum  temperatures  may 
not  be  more  than  five  degrees  apart.  On  the  other  hand, 
a  few  species  are  known  which  can  grow  anywhere  from 
15  to  68°. 

The  above  instances,  a  narrow  limit  of  five  degrees 
and  a  very  wide  limit  of  fifty  degrees,  may  be  considered  as 
exceptional.  In  general  it  may  be  said  that  bacteria  do 
not  grow,  or  but  very  poorly,  below  10°  and  above  40°. 

The  optimum  temperature  should  especially  be  sought 
for  when  cultivating  bacteria.  It  may  be  said  to  vary  with 
each  individual  species.  In  the  case  of  the  parasitic  bac- 
teria, those  which  grow  in  the  living  body,  the  temperature 
of  the  host  may  be  considered  as  best  adapted  for  their 
development.  Consequently  the  optimum  temperature  for 
the  parasitic  group  of  bacteria  may  be  placed  at  35  to  40°. 
On  the  other  hand,  the  saprophytic  bacteria,  those  which 
grow  in  nature  on  dead  matter,  find  the  warm  summer  tem- 
perature to  be  most  favorable  for  their  growth.  As  is  well 
known,  fermentative  and  putrefactive  changes  are  favored 
by  a  warm,  and  retarded  by  a  low  temperature.  Hence  the 
optimum  temperature  for  saprophytic  bacteria  may  be  said 
to  range  from  25  to  30°.  From  what  has  been  said,  it  is 
evident  that,  as  a  rule,  pathogenic  bacteria  should  be  cul- 
tivated in  an  incubator  at,  or  near,  the  temperature  of  the 
body  (37.5°);  whereas,  non-pathogenic  bacteria  are  grown 
best  at  the  ordinary  room  temperature. 


72  BACTERIOLOGY. 

Although,  as  a  general  rule,  bacteria  do  not  grow  below 
10°,  yet  a  number  of  notable  exceptions  are  known.  Sev- 
eral phosphorescing-  bacteria  grow  and  emit  light  even  at 
0°,  the  temperature  of  melting  ice.  Nearly  a  score  of 
organisms  have  been  studied  which  can  multiply  at,  or  near, 
the  freezing-point.  As  is  well  known,  meat  kept  in  cold 
storage  may  appear  perfectly  normal,  but  when  taken  out 
of  storage  it  will  decompose  much  more  rapidly  than 
ordinary  fresh  meat.  This  difference  is  due  to  the  fact 
that  the  bacteria  in  the  preserved  meat  slowly  multiply,  in 
spite  of  the  prevailing  low  temperature. 

The  temperature  of  40°  is  usually  considered  as  the 
the  maximum  temperature  for  the  growth  of  bacteria.  The 
majority  of  bacteria  cannot  grow  above  this  limit.  More- 
over, a  temperature  of  45  to  50°  will  rapidly  weaken,  and 
eventually  kill  all  the  ordinary  forms  of  bacteria.  There 
are,  however,  certain  bacteria  which  thrive  exceptionally 
well  at  such  abnormal  temperatures.  Indeed,  some  forty 
or  more  bacterial  species  have  been  isolated  which  grow  at 
58  to  60°.  A  few  species  have  been  observed  to  multiply 
at  68°  and  even  at  74°. 

The  bacteria  which  can  thus  grow  at  unusually  high 
temperatures  are  non-pathogenic,  and,  are  designated  as 
thermophilous.  When  it  is  remembered  that  the  ordinary 
vegetating  bacteria  are  promptly  killed,  in  a  few  minutes, 
by  a  temperature  of  60 — 70°  the  existence  of  this  group  of 
organisms  will  appear  all  the  more  remarkable.  The 
albumin  which  is  present  in  the  white  of  an  egg,  or  in  blood 
serum  will  coagulate  at  a  temperature  of  65 — 70°.  The 
destruction  of  bacteria  by  this  temperature  is  due,  there- 
fore, to  the  coagulation  of  the  albuminous  constituents  of 
the  protoplasm.  Evidently,  the  thermophilous  bacteria 
possess  a  markedly  different  chemical  composition. 

This  interesting  group  of  organisms  is  chiefly  repre- 
sented by  bacilli.  Only  a  few  micrococci  have  been  found 
to  grow  under  these  conditions.  The  thermophilous  bac- 


THE   ENVIRONMENT   OF   BACTERIA.  73 

teria  have  been  especially  isolated  from  garden  soiL 
Strange  to  say,  some  have  been  found  in  the  soil,  even  at  a 
depth  of  12  feet.  They  are  present  in  the  dust  of  rooms 
and  a  few  have  been  obtained  from  water,  especially  in  the 
case  of  hot  springs. 

In  the  latter  instance  the  necessary  temperature  is  fur- 
nished by  the  hot  water  It  is  more  difficult,  however,  to 
understand  how  the  organisms  of  this  group  can  obtain 
their  required  high  temperature  when  present  in  the  soil. 
The  heat  of  the  sun  will  not  warm  up  the  soil  to  this  extent. 
A  considerable  amount  of  heat  may  be  generated  by  the 
fermentative  changes  that  occur  on  the  surface  of  the 
earth.  The  heat  from  this  source  may  at  times  favor  the 
development  of  these  organisms.  It  has  been  shown  that 
these  bacteria,  under  anaerobic  conditions,  will  grow  at 
considerably  lower  temperatures  than  when  cultivated  in 
the  presence  of  air.  Thus,  certain  species  which  required 
a  temperature  of  50 — 70°  under  aerobic  conditions  could  be 
grown  as  anaerobes  at  34 — 44°.  The  anaerobic  conditions, 
as  shown  on  p.  70,  are  readily  supplied  in  the  soil  as  a 
result  of  microbic  associations.  This  explains  very  satis- 
factorily how  the  thermophilous  bacteria  can  live  in  nature. 
The  growth  of  these  organisms  at  temperatures  which  are 
rapidly  fatal  to  ordinary  bacteria  can  only  be  due  to  the 
presence  of  difficultly  coagulable  proteins. 

As  shown  above,  a  temperature  of  0°,  or  less,  stops  the 
multiplication  of  bacteria.  These  organisms,  however, 
may  be  exposed  to  extreme  cold  without  loss  of  vitality. 
In  the  case  of  bacteria,  cold  arrests  the  functions  of  pro- 
toplasm but  does  not  destroy  its  vitality.  This  is  true  not 
only  of  the  spore  condition  but  also  of  the  vegetating  form. 
The  typhoid  bacillus  may  remain  frozen  in  ice  for  months 
and  yet  be,  apparently,  uninjured.  The  weakest  individu- 
als, of  course,  will  die  out  first.  Moreover,  some  species 
are  more  susceptible  to  cold  than  others.  Alternate  freez- 


74  BACTERIOLOGY. 

ing  and  thawing-  tends  to  destroy  bacteria.  It  is  evident, 
therefore  that  cold  cannot  be  employed  for  the  purpose  of 
destroying-  bacteria. 

The  action  of  heat  is  very  different  from  that  of  cold. 
Thus,  many  pathogenic  bacteria  when  grown  at  41 — 43°  for 
some  time  become  so  weakened  that  they  are  unable  to 
grow  in  the  living-  body.  In  other  words,  they  become  at- 
tenuated. When  this  abnormal  condition  persists  it  eventu- 
ally causes  the  death  of  the  org-anism. 

The  higher  temperatures  act  in  a  similar  way.  They 
first  weaken  and  then  destroy  the  organism.  A  tempera- 
ture of  60 — 70°  will  destroy  the  ordinary  vegetating  bac- 
teria in  a  few  minutes.  In  such  cases  the  heat  probably 
causes  a  coag-ulation  of  the  protoplasm.  As  pointed  out 
on  p.  56,  spores  may  resist  the  action  of  steam-heat  for 
some  minutes,  and  even  for  some  hours,  whereas  the  vege- 
tating form  is  destroyed  instantaneously.  Heat  coagula- 
tion results  in  death  because  of  the  marked  alteration  in 
the  protein  constituents  of  the  cell.  In  cold  coagulation, 
on  the  other  hand,  the  water  within  the  cell  is  frozen  but 
the  protoplasm  is  not  altered.  It  is  evident  from  what  has 
been  said  above,  that  cold  acts  as  an  antiseptic,  whereas 
heat  is  a  germicide. 

When  cultivating  bacteria  it  is  very  important  that  the 
temperature  at  which  they  develop  shall  be  as  nearly  con- 
stant as  possible.  In  the  case  of  pathogenic  bacteria  this 
condition  is  usually  observed,  inasmuch  as  the  organisms 
are  grown  in  an  incubator  which  maintains  a  fairly  constant 
temperature.  When  saprophytic  bacteria  are  cultivated, 
or  whenever  g-elatin  is  employed,  this  is  done  at  the  so- 
called  room  temperature,  which  may  vary  considerably. 
At  night  it  may  be  10  or  15°  below  the  temperature  that 
prevails  during  the  day.  The  thermal  oscillations  of  this 
kind,  alternately  hastening  and  checking  the  growth  of 
bacteria,  are  not  without  effect  upon  the  form  of  the  organ- 
isms, of  the  colonies  and  upon  other  cultural  character- 


THE  ENVIRONMENT  OF   BACTERIA.  75 

istics,  such  as  pigment  production,  thickness  of  the  growth, 
etc.  A  constant  low  temperature  of  16 — 18°  is  as  desirable 
as  a  constant  high  temperature  in  an  incubator.  It  can 
readily  be  obtained  by  the  apparatus  shown  in  Fig.  33, 
Chapter  VII,  or  by  a  properly  constructed  ice-chest. 

Light. 

The  higher  plants,  in  the  presence  of  sun-light,  are 
able  to  assimilate  carbonic  acid.  The  relatively  few, 
colored  bacteria  are  likewise  favored  by  an  exposure  to 
sun-light.  Apart  from  these  few  organisms  it  may  be  said 
that  direct  sun-light  exerts  an  unfavorable  action  on  all 
bacteria.  Naturally,  some  species  will  be  less  affected 
than  others.  Moreover,  the  vegetating  form  will,  as  a 
rule,  be  more  easily  destroyed  than  the  spore  form.  A  few 
hours'  exposure  to  the  direct  sun  usually  destroys  vegetat- 
ing bacteria. 

The  action  of  sun-light  may  be  exerted  upon  the 
medium  as  well  as  upon  the  organism.  This  is  seen  in  the  fact 
that  sterile  bouillon,  or  urine,  after  an  insolation  of  some 
hours,  will  not  permit  a  development  of  certain  organisms. 
The  change  that  has  taken  place  in  the  soil  seems  to  inhibit 
the  germination  of  spores  more  readily  than  a  multiplica- 
tion of  the  organism  itself.  Clearly,  therefore,  a  pro- 
longed exposure  of  a  given  medium  to  sun-light  changes,  in 
some  way,  its  chemical  composition.  Similar  alterations 
will  be  met  with  when  strongly  alkaline  media  are  heated 
in  an  autoclave  for  some  time  at  120°.  The  action  of  sun- 
light, or  of  heat,  may  cause  an  oxidation  of  the  fats  or 
sugars  present  giving  rise  to  acid  products  which  change 
the  reaction  of  the  medium  and  consequently  render  it  use- 
less. Moreover,  oxidation  products  may  form,  like  formic 
acid,  or  formaldehyde,  which  exert  a  marked  antiseptic 
action.  Apparently  the  most  important  antiseptic  that 


76  BACTERIOLOGY. 

may  be  formed  in  the  presence  of  sun-light  is  hydrogen 
peroxide.  Insolated  urines  not  only  become  sterile  but 
remain  so,  as  a  result  of  the  formation  of  this  substance. 

The  action  of  direct  sun-light  is  especially  marked  in 
the  presence  of  air.  It  must  not,  however,  be  inferred  that 
oxygen  is  necessary.  Insolation  will  kill  bacteria  which 
are  kept  in  a  vacuum,  though  more  slowly  than  if  air  was 
present.  Oxygen,  therefore,  favors  or  assists  the  action 
of  sun-light. 

Diffuse  sun-light  has  very  little  or  no  action  on  ordinary 
bacteria.  Some  pathogenic  bacteria,  like  the  tubercle 
bacillus,  are  weakened  by  mere  exposure  to  day-light.  By 
far  the  best  results  are  obtained  when  bacteria  are  grown 
in  the  dark. 

Sun-light,  the  same  as  heat,  first  weakens  or  attenuates 
the  organism  and  finally  destroys  it.  Thus,  any  one  of  the 
bright  pigment-producing  bacteria  on  exposure  to  sun- 
light becomes  so  altered  physiologically  that  it  will  no  longer 
produce  a  pigment.  Colorless  varieties  can  thus  be  readily 
obtained.  In  the  case  of  disease-producing  bacteria  the 
weakened  condition  is  seen  in  the  fact  that  the  insolated 
organisms  will  no  longer  kill  animals. 


High  Pressure. 

When  bacteria  are  grown  under  a  high  pressure  they 
develop  under  unfavorable  conditions.  A  weakening  or 
attenuation  results  especially  if  a  number  of  successive 
generations  are  made  under  such  conditions.  Thus,  the 
anthrax  bacillus,  when  grown  for  several  generations  under 
a  pressure  of  9  atmospheres,  became  so  attenuated  that  it 
had  no  effect  upon  the  most  susceptible  animal,  the  white 
mouse.  That  bacteria  can  live  under  considerable  pressure 
is  seen  in  the  fact  that  certain  species  are  known  to  grow 
on  the  ocean's  bottom  at  a  depth  of  several  thousand  feet. 


THE   ENVIRONMENT   OF'  BACTERIA.  77 

Moreover,  direct  experiment  has  shown  that  many  bacteria 
can  multiply  under  a  pressure  of  several  hundred  atmos- 
pheres. Of  course  a  pressure  of  several  hundred  atmos- 
pheres to  the  square  inch  becomes  reduced,  owing-  to  the 
extremely  small  size  of  bacteria,  to  but  a  few  milligrams 
for  each  individual  cell. 


Electricity. 

It  is  very  difficult  to  demonstrate  a  direct  action  of 
electricity  on  bacteria.  This  is  due  to  the  fact  that  an 
electric  current  induces  c'hemical  and  physical  changes  in 
the  liquid.  The  chemical  changes  are  especially  marked 
in  solutions  of  various  salts.  Thus,  if  common  salt  is 
present  it  may  not  only  decompose  into  acid  and  alkali  but 
it  may  also  give  rise  to  free  chlorine  and  to  hypochlorites. 
The  bacteria  suspended  in  a  liquid  containing- -salt  may  be 
destroyed  as  the  result  of  the  passag-e  of  an  electric  cur- 
rent, but  the  immediate  cause  of  death  is  the  formation  of  the 
germicidal  products  mentioned  above.  Moreover,  hydrogen 
peroxide,  and  especially  ozone,  may  form  and  destroy  the 
bacteria  present.  The  purification  of  polluted  river- water 
by  means  of  ozone  g-enerated  by  powerful  currents  of  elec- 
tricity has  been  carried  on  successfully. 

In  addition  to  the  chemical  changes  caused  by  an 
electric  current  a  considerable  rise  in  temperature  may  be 
observed.  Obviously,  the  heat  thus  generated  will  not  be 
without  effect  upon  the  organisms  in  suspension. 

It  is  not  possible  to  overcome,  or  to  do  away  with  the 
action  of  the  chemical  products  and  of  heat  when  studying 
the  action  of  an  electric  current.  It  would  seem  as  if  the 
latter  possessed  little  or  no  direct  action.  A  feeble  gal- 
vanic current  has  little  or  no  effect,  whereas  a  strong  cur- 
rent, especially  if  allowed  to  act  for  some  hours,  will  destroy 
the  organisms  present.  That  this  result  is  due  to  an  alter- 


78  BACTERIOLOGY. 

ation  of  the  medium  and  the  presence  of  germicidal  sub- 
stances can  be  easily  demonstrated.  Such  a  medium  when 
subsequently  inoculated  with  a  fresh  culture  will  remain 
sterile. 

The  effect  of  X-rays  on  bacteria  has  been  the  subject 
of  considerable  study.  These  rays,  as  in  the  case  of  the 
electric  current,  can  be  said  to  be  without  any  direct  action. 

Chemotaxis. 


The  lower  forms  of  life  exhibit  a  singular  behavior  with 
reference  to  certain  chemical  substances.  If,  for  instance, 
a  minute  capillary  tube  is  partly  filled  with  a  solution  of 
pepton  or  meat  extract  and  the  end  of  the  tube  is  placed  in 
a  drop  of  water  which  has  been  inoculated  with  a  small 
number  of  motile  bacteria  it  will  be  found  that,  in  a  few 
minutes,  all  the  bacteria  will  have  gathered  around  the 
tube.  This  attraction  of  the  bacteria  by  the  chemical  sub- 
stance in  the  tube  is  known  as  chemotaxis.  The  various 
chemical  substances  will  show  different  degrees  of  attrac- 
tion. Moreover,  the  concentration  of  the  solution  has 
much  to  do  with  the  result.  Other  compounds,  such  as 
acids,  alkalis  and  alcohol  do  not  attract  bacteria  and,  in- 
deed, may  be  said  to  exert  a  repellant  action.  It  is  cus- 
tomary, therefore,  to  speak  of  positive  and  of  negative 
chemotaxis. 

The  phenomenon  of  chemotaxis,  which  was  first  ob- 
served on  higher  plants,  and  then  on  lower  forms,  has  been 
utilized  to  explain  the  behavior  of  the  cells  of  the  body  in 
the  presence  of  invading  bacteria.  It  should  be  remem- 
bered that  there  is  no  satisfactory  explanation  of  the 
phenomenon  itself. 


CHAPTER  V. 
THE  CHEMISTRY  OF  BACTERIA. 

All  living-  cells  take  in  food,  build  up  protoplasm  and 
throw  off  waste — or  metabolic  products.  In  a  multi-cellular 
organism,  such  as  the  higher  plant  or  animal,  it  is  not  pos- 
sible to  trace  each  of  the  various  chemical  compounds  which 
are  given  off  back  to  the  cell  which  made  them.  In  the  case 
of  the  uni-cellular  organisms  this  can,  as  a  rule,  be  easily 
done.  Although  a  number  of  different  species  may  be  grow- 
ing together  in  a  liquid  it  is  possible  to  separate  the  species 
from  one  another  and  to  obtain  thus  pure  cultures.  The  chem- 
ical products  of  each  species  can  then  be  studied,  unmixed 
with  the  products  of  other  organisms. 

A  large  number  of  bacterial  species  may  grow  upon  one 
and  the  same  nutrient  medium.  The  chemical  products,, 
however,  will  vary  with  each  species.  Thus,  one  organism 
growing  upon  a  given  medium  may  produce  an  acid  whereas 
another  species  growing  upon  the  same  medium  may  give 
rise  to  an  alkaline  reaction.  In  some  instances  differences 
of  this  kind  are  very  marked  and  are  utilized  for  the  pur- 
pose of  distinguishing  one  species  from  another.  Thus,  the 
colon  bacillus  produces  acid  and  gaseous  products  and  in- 
dol,  whereas  the  typhoid  bacillus,  which  otherwise  resem- 
bles the  former  very  much,  does  not  elaborate  either  one  of 
these  products. 

A  given  micro-organism  always  produces  a  considerable 
number  of  different  chemical  compounds.  Thus,  the  yeast 
plant  is  said  to  produce  alcohol,  but  it  must  not  be  inferred 
that  this  is  the  only  substance  which  it  elaborates.  On  the 
contrary  it  produces  gases,  several  kinds  of  alcohol,  a  num- 


80  BACTERIOLOGY. 

ber  of  acids,  ferments  and  the  like.  The  composition  of 
the  nutrient  medium  will,  of  course,  influence  the  kind  of 
products.  Thus,  grape-sugar  is  readily  fermented  by  the 
yeast-plant  yielding-  alcohol,  carbonic  acid  and  other  pro- 
ducts whereas  milk-sugar  is  not  affected. 

The  several  chemical  compounds  elaborated  by  a  given 
species  are  subject  to  variation  depending  upon  the  health 
and  vigor  of  the  organism.  If  the  latter  has  been  weakened 
by  an  abnormally  high  temperature,  by  sun-light,  or  by  pois- 
onous chemicals  it  will  be  found  that  this  change  in  the  phy- 
siological condition  of  the  cell  will  be  evidenced  by  altered 
waste-products.  Thus,  a  normal  species  may  give  rise  to  a 
bright-red  pigment  whereas  the  attenuated  or  weakened 
variety  may  yield  a  perfectly  colorless  growth.  Again,  a 
certain  organism  may  normally  curd  le  milk  in  a  few  hours,  but 
when  altered  it  will  do  so  very  slowly  or  not  at  all.  Numerous 
other  instances  might  be  cited  to  show  that  the  kind  of  pro- 
ducts formed  depends  not  only  upon  the  composition  of  the 
soil,  but  also  upon  the  physiological  condition  of  the  cell. 

In  the  study  of  the  chemistry  of  bacteria  and  allied 
forms  it  will  frequently  be  found  that  a  given  chemical 
compound  is  produced  by  several  distinct  species.  Alcohol 
is  said  to  be  the  product  of  the  yeast-plant.  More  than  a 
score  of  species  of  the  yeast-plant,  however,  are  known  to 
possess  the  power  of  making  alcohol.  One  species  may,  of 
course,  produce  a  much  larger  amount  than  another.  More- 
over, alcohol  is  produced  by  many  bacteria  and  by  moulds. 
What  has  been  said  of  alcohol  is  equally  true  of  acetic,  lac- 
tic, butyric  acids  and  other  compounds.  In  other  words, 
entirely  different  species  of  organisms  may  give  rise  to  a 
given  chemical  compound,  though  in  different  amounts.  It 
is  evident,  therefore,  that  alcoholic,  acetic,  etc.,  fermenta- 
tions are  not  necessarily  due  to  one  species. 

When  bacteria  grow  in  a  tube  or  flask  the  various  waste- 
products  which  they  elaborate  remain  in  the  liquid  in  direct 


THE    CHEMISTRY  OF    BACTERIA.  81 

contact  with  the  cells  that  produced  them.  The  accumula- 
tion of  waste-products  eventually  stops  the  growth  of  the 
organism,  and  as  pointed  out  on  p.  50,  in  many  species  it 
will  induce  spore  formation.  Prolonged  contact  with  these 
chemical  products  is  injurious  and  may  result  in  the  death 
of  the  organism.  If  the  bacteria  are  grown  in  a  tube,  the- 
walls  of  which  are  permeable,  as  in  the  case  of  a  collodium 
sac,  the  waste-products  will  diffuse  through  the  wall  into 
the  surrounding  liquid,  and,  as  a  result,  growth  will  continue 
as  long  as  nutrient  substances  are  present.  A  similar  diffu- 
sion, or  rather  dilution,  enables  these  organisms  to  grow, 
more  or  less  continuously,  in  various  waters. 

The  accumulated  waste-products  under  ordinary  experi- 
mental conditions  soon  stop  the  growth  and  multiplication 
of  the  germs  present.  Inasmuch  as  the  chemical  products 
of  one  organism  are  more  or  less  different  from  those  of  an- 
other, it  will  be  evident  that  in  some  instances  the  inhibition 
will  be  more  marked  than  in  others.  Thus,  in  lactic  acid 
fermentation  the  process  stops  when  about  0.8  per  cent,  of 
free  lactic  acid  has  formed.  On  the  other  hand,  in  acetic 
acid  fermentation  the  growth  may  not  be  inhibited  by  10 
per  cent,  of  the  free  acid.  Similarly,  the  yeast-plant  may 
continue  to  grow  until  about  15  per  cent,  of  alcohol  has  ac- 
cumulated. Certain  bacteria  give  rise  to  phenol  or  carbolic 
acid  which  is  a  well  marked  antiseptic  even  when  consider- 
ably diluted. 

Although  bacteria  are  small  in  size  and  simple  in  struc- 
ture they,  nevertheless,  elaborate  a  great  variety  of  chem- 
ical compounds.  The  changes  which  these  minute  organ- 
isms induce  are  exceedingly  complex.  In  the  first  place  it 
must  be  borne  in  mind  that  the  cell  builds  up  its  protoplasm 
and  other  cell  constituents  out  of  the  food  supplied.  Such 
a  chemical  change  is  obviously  carried  on  within  the  cell. 
It  is  intracellular  and  synthetic.  The  bacterial  proteins, 
toxins  and  ferments  are  undoubtedly  formed  in  this  way. 

G 


82  BACTERIOLOGY. 

On  the  other  hand,  the  waste-products  are  formed  in  the 
cell  and  are  excreted.  They  result  from  a  tearing-down,  or 
cleavage  of  more  complex  bodies  and,  therefore,  are  to  be 
considered  as  analytic  products. 

Inasmuch  as  the  compounds  mentioned  are  formed 
within  the  cell  they  may  be  designated  as  primary  products. 
These  should  be  distinguished,  as  far  as  possible,  from  .the 
secondary  products  which  are  formed  wholly  on  the  outside 
of  the  cell.  Thus,  the  ferment  produced  within  the  cell  may 
be  eliminated  and  may  then  act  upon  suitable  material  on 
the  outside.  The  pepsin  of  the  gastric  juice  is  secreted  by 
certain  cells  of  the  stomach  and,  whether  in  the  stomach  or 
in  a  test-tube,  it  will  digest  or  split  up  albumin  into  simpler 
bodies  such  as  albumose,  or  pepton.  In  like  manner,  the  fer- 
ment produced  by  bacteria  may  act  upon  various  substances 
and  give  rise  to  cleavage  products.  Again,  certain  bacteria 
produce  compounds  which  unite  with  the  oxygen  of  the  air 
to  form  bright-colored  pigments.  In  either  case,  the  pro- 
ducts are  not  made  directly  by  the  cell  and  for  this  reason 
they  may  be  spoken  of  as  secondary. 

Bacterial  proteins. — The  albuminous  substances  which 
are  formed  within  the  cell,  and  which  constitute  an  integral 
part  of  the  cell  protoplasm,  are  known  as  the  bacterial 
cellular  proteins.  Very  little  is  known  in  regard  to  their 
exact  chemical  composition.  The  cellular  proteins  of  some 
bacteria  possess  a  marked  action  upon  the  animal  body. 
They  may  cause  suppuration,  or  may  possess  a  decided 
poisonous  action. 

Secondary  bacterial  proteins  are  formed  outside  of  the 
cell,  in  the  culture  medium,  by  the  action  of  the  soluble 
ferments  secreted  by  the  organisms.  Various  albuminous 
substances  may  consequently  be  found  in  the  liquid  in 
which  bacteria  are  growing.  Thus,  the  coagulated  white 
of  an  egg,  or  ordinary  blood-fibrin,  may  be  dissolved  and 
changed  to  an  albumose.  If  the  ferment  is  very  active  the 


THE    CHEMISTRY  OF    BACTERIA. 

albumose  may  in  turn  be  split  up  into  pepton.  It  is  evident, 
then,  that  a  bacterial  liquid  may  contain  in  solution,  albu- 
min, globulin,  albumoses,  or  even  pepton.  These  protein 
substances  will  be  precipitated  from  such  culture  fluids  by 
the  addition  of  several  volumes  of  absolute  alcohol.  If 
harmless,  non-poisonous  bacteria  were  cultivated  in  the- 
liquid  the  precipitated  proteins  will  produce  no  effect  when 
injected  into  an  animal.  On  the  other  hand,  in  the  case  of 
injurious,  disease-producing-  bacteria  the  precipitate  will  be 
highly  poisonous.  In  this  way  a  tox-albumin  was  obtained 
from  cultures  of  the  diphtheria  bacillus;  a  tox-albumose 
from  those  of  the  anthrax  bacillus,  and  a  toxo-pepton  from 
those  of  the  cholera  vibrio. 

The  fact  that  the  precipitated  protein  is  poisonous  does 
not  prove  that  the  protein  itself  is  actually  poisonous. 
There  is  every  reason  to  believe  that  the  protein  substances 
present  in  a  bacterial  liquid  are  non-poisonous.  When, 
however,  they  are  thrown  out  of  solution  they  mechanically 
drag  down  the  real  bacterial  poison  and,  as  a  result,  the 
precipitate  is  toxic.  If  a  precipitate  of  calcium  phosphate 
or  of  aluminum  hydrate  is  produced  in  such  a  liquid  it  will 
be  likewise  poisonous  for  the  'reason  given. 

Although  the  "  toxalbumins,"  etc.,  are  not  the  real 
bacterial  poisons  as  was  at  one  time  supposed,  it  neverthe- 
less remains  true  that  certain  albuminous  substances, 
elaborated  by  higher  plants  and  animals,  may  be  intensely 
poisonous.  There  is  reason  to  believe  that  the  venom  of 
serpents  owes  its  marked  poisonous  action  to  certain  pro- 
teins, such  as  pepton  and  globulin.  Again,  the  jequirity 
seed  contains  an  albumose  body,  known  as  abrin,  which  is 
very  toxic.  When  injected  intravenously,  abrin  is  fatal  in  a 
dose  of  0.00001  g.  per  kg.  body  weight.  In  other  words,  1  g. 
of  abrin  is  capable  of  killing  200,000  guinea-pigs,  each  weigh- 
ing one  pound.  The  calculated  fatal  dose  for  a  man  weighing 
130  pounds  would  be  riv  grain.  A  somewhat  less  poisonous 
albumose,  ricin,  has  been  obtained  from  the  castor  bean. 


84  BACTERIOLOGY. 

Toxins, — It.  has  been  pointed  out  (p.  79)  that  a  given 
micro-organism  produces  a  considerable  number  of  chemical 
compounds.  •  It  is  possible  for  several  of  these  products  to 
be  more  or  less  poisonous.  In  the  case  of  the  disease- 
producing"  bacteria  each  one  elaborates  a  highly  toxic  sub- 
stance, which  has  been  designated  by  the  term  toxin.  It 
is  certain  that  these  toxins,  the  specific  bacterial  poisons, 
are  made  within  the  cell,  by  synthetic  processes.  Thus  far, 
no  one  has  succeeded  in  isolating  toxins  in  a  strictly  pure 
condition,  and  consequently  their  chemical  composition  is 
undetermined.  While  it  is  not  known  what  the  toxins  are,  it 
nevertheless  has  been  definitely  shown  that  they  are  not  basic 
substances  or  ptomains,  and  that  they  are  not  proteins. 

Obviously  the  toxin  of  the  diphtheria  bacillus  is  differ- 
ent from  that  of  tetanus,  or  of  cholera.  Each  pathogenic 
organism,  therefore,  makes  a  toxin  which  is  characteristic 
for  that  particular  species.  In  the  case  of  the  tetanus  and 
diphtheria  bacilli,  the  specific  toxins  readily  pass  out  of 
the  cell  into  the  surrounding  liquid.  Consequently,  the 
filtered  cultures  of  such  organisms  may  be  intensely  poison- 
ous. It  is  possible,  for  instance,  to  obtain  a  filtered  diph- 
theria culture  which  will  kill  a  guinea-pig  in  a  dose  of 
0.002  c.  c.,  or  even  less.  The  toxin,  of  course,  makes  up 
probably  only  a  small  fraction  of  one  per  cent,  of  this  dose. 
The  purified  tetanus  toxin  was  fatal  to  a  15  g.  mouse  in  a 
dose  of  0.000,000,05  g.  In  other  words,  a  mouse  weighing 
half  an  ounce  is  killed  by  Trsiwcr  of  a  grain  of  the  purified 
toxin.  The  calculated  fatal  dose  for  a  man  weighing  155 
pounds  is  gfo  grain.  It  is  evident  that  these  toxins  are  by 
far  the  most  powerful  poisons  known  to  man. 

In  the  instances  cited,  the  specific  toxin  readily  passes 
out  of  the  cell  into  the  surrounding  liquid.  This,  however, 
is  not  always  the  -case.  As  a  matter  of  fact,  the  toxin  is 
usually  retained  within  the  cell  and  consequently  such  cul- 
tures, when  filtered,  are  but  very  slightly  poisonous.  The 
toxin  which  is  stored  up  within  the  organism  may  be 


THE    CHEMISTRY  OF    BACTERIA.  85 

liberated  on  the  death  of  the  cell.  Moreover,  the  outward 
diffusion  of  the  toxin  is  favored  by  certain  conditions  which 
are,  as  yet,  but  imperfectly  understood. 

The  toxins  are  very  unstable  chemical  compounds. 
They  are  weakened,  or  destroyed  by  heat,  light,  exposure 
to  oxygen  and  by  various  chemical  reagents. 

Ferments. — As  a  rule,  a  substance  in  order  to  be  util- 
ized as  a  food  must  be  brought  into  solution.  The  starch 
present  in  bread,  or  in  the  potato,  is  insoluble  in  water  and 
consequently  would  be  valueless  to  an  animal  if  the  latter 
did  not  possess  some  means  of  splitting  up  the  starch 
molecule  and  thus  bringing  its  constituents  into  solution. 
The  saliva  and  the  pancreatic  secretion  contain  soluble, 
starch-splitting  ferments  which  act  upon  starch  and  con- 
vert it  into  sugar.  The  sugar  can  now  be  readily  absorbed 
and  utilized  by  the  animal  organism.  A  hard  boiled  egg, 
or  a  piece  of  meat,  consists  chiefly  of  protein  or  albuminous 
matter,  which  is  practically  insoluble.  As  long  as  it  is  in 
this  insoluble  condition  it  cannot  be  absorbed  by  the  living 
organism  and  utilized  as  food.  It  is  well  known,  however, 
that  the  hardest  egg  and  toughest  meat  can  be  readily  dis- 
solved by  the  aid  of  the  protein-splitting  ferments  con- 
tained in  the  gastric  and  pancreatic  secretions.  The  fats 
can  be  absorbed  directly,  when  in  a  very  finely  divided 
condition.  The  formation  of  an  emulsion  is  necessary  and 
this  is  brought  about  by  the  fat-splitting  ferment  of  the 
pancreatic  juice. 

It  is  evident,  then,  that  in  the  case  of  the  animal  the 
cells  of  the  salivary  gland,  of  the  stomach,  and  of  the  pan- 
creas produces  certain  soluble  substances  known  as  fer- 
ments or  enzymes.  These  ferments  are  necessary  to  the 
preparation  of  the  food  for  absorption  and  without  their 
aid,  it  is  safe  to  say,  the  animal  could  not  exist.  What  is 
true  of  the  higher  form  of  life  is  equally  true  of  lower  forms. 
When  bacteria  are  planted  on  coagulated  egg  albumin,  or 


86  BACTERIOLOGY. 

on  blood-serum  they  cannot  utilize  this  material  as  food 
unless  some  of  it  is  first  dissolved,  and  in  this  condition  only 
can  it  be  absorbed  through  the  cell-wall.  Bacteria  must 
therefore  produce  proteolytic  ferments  analogous  to  those 
made  by  the  stomach  and  by  the  pancreatic  gland. 

Many  bacteria  are  characterized  by  the  rapidity  with 
which  they  digest  or  dissolve  albuminous  matter.  The  liq- 
uefaction of  gelatin  by  certain  bacteria  is  due  to  the  forma- 
tion of  peptonizing  ferments.  Many  bacteria  do  not  liquefy 
gelatin  and  this  would  seem  to  indicate  the  absence  of  this 
class  of  enzymes.  There  is  reason  to  believe  that  even 
these  organisms  produce  such  ferments  which,  however,  do 
not  readily  diffuse  into  the  surrounding  medium.  The 
tubercle  and  typhoid  bacilli  do  not  peptonize  coagulated 
blood-serum  and  yet  the  cell  contents  in  each  case  will  show 
a  distinct,  though  slow  proteolytic  action.  It  has  been  shown 
above  (p.  84)  that  certain  bacteria  elaborate  toxins,  which 
in  some  species,  may  readily  pass  out  of  the  cell,  whereas 
in  others  they  are  apparently  retained  within  the  cell.  The 
same  is  apparently  true  of  the  bacterial  ferments,  which  in 
some  cases  easily  diffuse  outward  (liquefying  bacteria), 
whereas  in  others  they  tend  to  remain  within  the  cell. 

Bacteria,  clearly,  cannot  absorb  the  insoluble  starch. 
Like  the  animal  cell  they  can  make  use  of  starch  as  food 
only  after  it  has  been  dissolved  and  converted  into  sugar. 
They,  therefore,  secrete  starch-splitting  or  amylolytic  fer- 
ments. In  some  bacteria  this  action  on  starch  is  quite 
marked,  whereas  in  others  it  is  apparently  absent.  The 
action  of  bacteria  on  fats,  likewise,  is  largely  due  to  the 
presence  of  a  soluble  ferment. 

It  has  been  customary  to  divide  the  so-called  ferments 
into  two  groups: 

1.)     Organized,  formed,  living,  or  insoluble  ferments; 
2.)     Unorganized,  formless,  non-living,    or    soluble  fer- 
ments. 


THE    CHEMISTRY  OF    BACTERIA.  87 

The  yeasts,  bacteria  and  moulds  were  designated  as  or- 
ganized ferments  because  they  possess  a  cell  structure, 
and  act  on  organic  matter  like  the  unorganized  or  soluble 
ferments  secreted  in  the  animal  body.  There  can  be  no 
doubt  but  that  the  ferment  action  of  living  cells,  such  as 
yeasts,  bacteria,  etc.,  is  due  to  soluble  ferments  or  enzymes 
which  diffuse  outward,  more  or  less  readily.  The  uni-cellular 
organism  requires  soluble  ferments  or  enzymes,  just  as  do 
other  forms  of  life.  The  sprouting  seed  contains  such  fer- 
ments which  are  necessary  to  the  nourishment  of  the  em- 
bryo, enabling  it  to  utilize  the  starch  and  other  stored  up 
foods.  Malt,  for  instance,  contains  not  only  an  amylolytic 
ferment,  diastase,  but  also  a  proteolytic  ferment.  The  yeast 
cell  contains  a  ferment,  which  changes  glucose  into  alcohol 
and  carbonic  acid.  It  also  contains  a  peptonizing  ferment. 
Papain,  a  vegetable  ferment,  shows  a  marked  proteolytic 
action. 

Soluble  ferments  or  enzymes  are,  therefore,  produced  by 
plants  as  well  as  by  animals  and  are  primarily  necessary  to 
the  absorption  of  certain  foods. 

It  will  be  seen  from  the  above  that  the  soluble  ferments 
or  enzymes  differ  in  their  action.  The  more  common  en- 
zymes may  be  grouped  or  designated  according  to  their 
characteristic  behavior,  as: 

Diastatic  or  amylolytic, 

Peptonizing  or  proteolytic, 

Fat-splitting, 

Inverting, 

Rennet-like. 

The  diastatic  ferments  are  designated  thus  because  they 
resemble  in  their  action  the  well  known  ferment  of  malt,— 
diastase.  Like  diastase  they  split  up  starch,  first  into  dex- 
trin, and  this  in  turn  is  changed  into  sugar.  The  saliva  and 
the  pancreatic  juice  contain  amylolytic  ferments.  These 
compounds  are  frequently  met  with  in  higher  plants,  cspe- 


88  BACTERIOLOGY. 

cially  in  sprouting-  seeds.  Malt,  or  sprouting-  barley,  is 
rich  in  diastase.  Similar  ferments  are  elaborated  by 
bacteria. 

The  proteolytic  enzymes  act  on  albuminous  substances 
and  convert  these  into  albumose  and  the  latter  into  pepton. 
The  pepsin  of  the  gastric  juice  and  the  trypsin  of  the  pan- 
creatic secretion  will  serve  as  types  of  this  class  of  ferments. 
As  indicated  above,  similar  enzymes  are  met  with  in  the 
higher  as  well  as  in  the  lower  forms  of  plant-life.  The 
liquefaction  or  the  peptonizing  of  gelatin  is  due  to  similar 
ferments. 

The  fat-splitting  ferment  breaks  up  neutral  fats  into 
glycerin  and  fatty  acids.  A  ferment  cf  this  kind  is  pres- 
ent in  the  pancreatic  secretion.  Many  bacteria  possess  a 
similar  action  which  may  be  carried  on  in  the  intestines,  or 
otherwise.  The  rancidity  of  fats,  especially  butter-fat,  is 
frequently  due  to  such  organisms. 

Inverting  ferments  change  cane-sugar  into  glucose. 
They  are  elaborated  by  plants  as  well  as  by  animals. 

Rennet,  as  is  well  known,  coagulates  or  curdles  milk. 
The  gastric  secretion  of  all  vertebrate  animals  contains  a 
rennet  ferment.  The  mucous  membrane  of  the  calf's  stom- 
ach, is  utilized  for  this  reason  in  the  preparation  of  curd 
or  cheese.  A  rennet-like  action  is  exhibited  by  many 
bacteria. 

Ptomains. — These  are  organic,  basic  products  resulting 
from  the  action  of  bacteria  on  albuminous  matter.  They  all 
contain  C,  H,  and  N,  and  some  contain  O.  They  are  alka- 
line substances  which,  when  neutralized  by  acids,  yield  crys- 
tallizable  salts.  Most  of  the  ptomains  are  not  poisonous, 
or  but  very  feebly  so.  Only  a  few  can  be  said  to  be  very 
toxic. 

The  discovery  of,  more  or  less,  poisonous  basic  substan- 
ces among  the  bacterial  products  led  to  the  belief  that  bac- 
teria produced  disease  by  giving  rise  to  ptomains.  This 


THE    CHEMISTRY  OF    BACTERIA.  89 

view  was  largely  based  upon  analogy.  Thus,  it  is  well 
known  that  many  of  the  poisonous  higher  plants  owe  their 
injurious  action  to  the  presence  of  the  so-called  vegetable 
alkaloids.  Strychnin,  morphin,  atropin,  nicotin,  etc,,  are 
examples  of  this  kind.  It  soon  became  evident,  however, 
that  the  ptomains  could  not  be  considered  as  the  chief, 
poisons  elaborated  by  bacteria.  In  the  first  place,  the 
amount  of  ptomains  present  in  a  filtered  bacterial  liquid  was 
too  small  to  account  for  the  intensely  poisonous  action  of 
such  a  liquid.  Moreover,  certain  bacteria,  like  the  diph- 
theria bacillus,  do  not  give  rise  to  ptomains.  Clearly,  there- 
fore, the  real  weapon  possessed  by  bacteria  is  of  a  different 
chemical  nature. 

It  has  already  been  shown  that  the  specific  poisons  of 
the  pathogenic  bacteria  are  known  as  toxins.  They  are 
certainly  not  basic  substances  or  ptomains,  and  they  are 
not  protein  compounds.  The  ptomains,  consequently,  are 
of  only  secondary  importance  as  factors  in  the  production 
of  disease.  They  are  simple,  waste-products  which  result 
from  the  breaking  down  of  albuminous  matter. 

Alkalis. — Many  bacteria  impart  an  alkaline  reaction  to 
the  medium  in  which  they  grow.  As  a  rule,  certain  nitro- 
genous substances  must  be  present  in  order  that  alkaline 
products  may  form.  The  protein  compounds  are  of  this 
kind.  In  the  bacterial  decomposition  of  urine  the  urea 
is  decomposed  into  ammonia  and  carbonic  acid.  Ammonia  is 
the  most  common  alkali  made  by  bacteria.  It  represents 
the  final,  inorganic  form  to  which  the  nitrogen  of  dead 
protoplasm  is  eventually  reduced.  An  alkaline  reaction 
may  result  from  the  oxidation  of  sodium  acetate  and 
similar  salts.  In  such  instances  sodium  carbonate  would 
form. 

Certain  organic  derivatives  of  ammonia  are  frequently 
met  with.  These  are  known  as  amines  and  result  from 
the  introduction  of  an  organic  group  into  the  ammonia 


90 


BACTERIOLOGY. 


molecule.      This   can   be   understood   from    the   following- 
formulae! 


/H 

N— H 

Ammonia. 


/H 

N— H 
XCH3 

Methylamin. 


/H 
N-CH3 

XCH3 

Di-methylamin. 


N-CH3 
XCH3 

Tri-methylamin. 


The  amines  possess  a  fish-  or  herring-like  odor.  Com- 
pounds of  this  kind  are  present  in  herring-brine  and  are 
frequently  formed  in  the  decomposition  of  protein  matter. 
Inasmuch  as  they  are  organic  and  basic  in  character  they 
belong  to  the  group  of  ptomains. 

Acids. — Products  of  this  kind  are  very  common.  They 
usually  result  from  the  oxidation  of  carbohydrates.  Thus, 
glucose  is  readily  split  up  into  acid  products  by  a  large 
number  of  bacteria.  One  organism  may  convert  it  into 
lactic  acid,  whereas  another  may  produce  butyric  acid. 
Hydrogen  sulphide,  an  acid  product,  will,  of  course,  be 
formed  only  out  of  certain  sulphur  containing  compounds, 
such  as  the  proteins.  These  compounds,  moreover,  may 
give  rise  to  various  fatty  acids. 

It  must  be  understood  that  a  given  organism  may  pro- 
duce acids  and  alkalis  at  the  same  time.  The  final  reaction 
in  that  case  will  depend  upon  which  of  these  compounds  is 
present  in  excess.  It  not  infrequently  happens  that  an 
alkaline  medium  turns  acid  and  then,  after  a  few  days, 
again  becomes  alkaline. 

The  acid  products  of  bacteria  are  very  numerous.  The 
fatty  acid  series  is  usually  represented  by  formic,  acetic, 
propionic  and  butyric  acids.  The  higher  fatty  acids,  such 
as  stearic  and  palmitic  acids,  will  form  in  the  decom- 
position of  proteins,  or  of  fats.  Lactic  acid  is  very  com- 
mon. Oxalic  and  succinic  acids  may  also  be  formed. 
Carbonic  acid  and  hydrogen  sulphide  are  likewise  to  be 


THE    CHEMISTRY   OF   BACTERIA.  91 

mentioned  among-  the  acid  products.  Amido-acids,  such 
as  leucin,  tyrosin,  etc.,  may  be  formed  in  protein  decom- 
position. 

The  fact  that  some  bacteria  readily  give  rise  to  acids, 
whereas  others  do  not,  can  be  made  use  of  for  the  purpose 
of  recognition.  The  reaction  can  best  be  observed  by_ 
coloring-  the  medium  with  some  litmus  solution.  When 
acids  are  produced  the  litmus  colored  medium  will  assume 
a  red  color,  otherwise  it  remains  blue.  The  bacillus  of 
tetanus,  for  example,  does  not  redden  glucose  g-elatin, 
whereas  that  of  malignant  edema  does.  Ag-ain,  the 
typhoid  bacillus  does  not  produce  an  acid  when  grown  on 
media  which  certain  milk-sug-ar,  whereas  the  colon  bacillus 
does. 

Alcohols. — In  the  decomposition  of  carbohydrates  by 
bacteria  several  alcohols  may  form,  but  the  amount  is 
usually  very  small.  Ethyl  and  butyl  alcohols  have  been 
frequently  met  with.  Glycerin  is  produced  in  small  quan- 
tity by  the  yeast-plant.  Phenol  or  carbolic  acid,  which  is 
liberated  in  the  putrefaction  of  proteins,  belongs  under 
this  head. 

Gases. — The  gaseous  products  of  bacterial  growth  are 
of  considerable  interest.  It  has  already  been  indicated  that 
the  different  bacteria  give  off,  or  exhale,  variable  amounts 
of  carbonic  acid.  It  may  happen  in  such  cases  that  no 
visible  evolution  of  gas  occurs.  Usually,  however,  the 
presence  of  bubbles  in  the  liquid  or  solid  media  indicates 
the  active  generation  of  gaseous  products.  An  increased 
formation  of  gas  is  observed  when  glucose  is  added  to  the 
culture  medium. 

The  carbohydrates,  in  general,  are  very  prone  to  give 
rise  to  gaseous  products  when  acted  upon  by  micro-organ- 
isms. Carbonic  acid  represents  the  final  form  to  which  all 
organic  substances  are  eventually  changed.  It  is,  conse- 


92  BACTERIOLOGY. 

quently,  a  very  common  product.  Marsh-gas  is  found 
wherever  cellulose  is  undergoing-  decomposition.  It  is 
sometimes  an  important  constituent  of  the  intestinal  gases. 
The  volatile  amine  compounds  have  been  already  referred 
to.  Carbon  monoxide  may  possibly  occur  among  the 
bacterial  products. 

Free  nitrogen  and  hydrogen  are  sometimes  met  with. 
Sulphur  occurs  frequently  in  the  gaseous  condition  as 
hydrogen  sulphide.  Phosphorus  may  likewise  appear  as 
phosphuretted  hydrogen.  It  is  evident,  therefore,  that 
proteins,  as  well  as  carbohydrates,  give  off  gaseous  decom- 
position products. 

The  formation  of  gas  bubbles  is  made  use  of  in  differ- 
entiating one  species  from  another.  Thus,  the  colon 
bacillus  produces  gas  in  abundance,  whereas  the  typhoid 
bacillus  does  not.  The  anaerobic  bacteria  possess  especial- 
ly marked  aerogenic  properties. 

As  a  result  of  abnormal  fermentations  in  the  stomach, 
or  in  the  intestines,  a  considerable  amount  of  gas  may  be 
produced.  Moreover,  in  certain  bacterial  diseases,  as 
symptomatic  anthrax,  a  marked  accumulation  of  gas  may 
occur  in  the  subcutaneous  tissue.  In  such  cases  a  distinct 
crackling  sensation  is  felt  when  the  fingers  are  passed  over 
the  skin.  An  interesting  pathological  phenomenon  is  met 
with  in  the  so-called  "foaming  liver."  On  the  cut  surface 
of  this  organ  gas  bubbles  may  appear  and  form  a  scum 
which  rapidly  increases  in  thickness.  When  this  foam  is 
removed  a  new  one  will  form.  This  peculiar  condition  is, 
of  course,  met  with  only  when  certain  markedly  aerogenic 
bacteria  are  present.  Inasmuch  as  the  gases  produced  fre- 
quently contain  marsh-gas  and  hydrogen  they  will  burn  on 
contact  with  a  lighted  match. 

The  various  bacteria  can  be  designated,  or  grouped, 
according  to  the  characteristic  action  which  they  induce. 


THE    CHEMISTRY  OF    BACTERIA.  93 


The  following  terms  are  frequently  employed  in  this  con- 
nection: 

Zymogenic,  or  fermentation  bacteria, 
Saprogenic,  or  putrefaction  bacteria, 
Chromogenic,  or  pigment  bacteria, 
Aerogenic,  or  gas  producing  bacteria  (p.  91), 
Photogenic,    or    light    producing,    phosphorescing 

bacteria, 

Toxicogenic,  or  poison  producing  bacteria, 
Pathogenic,  or  disease  producing  bacteria. 

It  must  not  be  supposed  from  the  above  grouping  of 
bacteria  that  a  given  species  is  necessarily  confined  or  lim- 
ited to  one  or  the  other  of  these  groups.  It  should  be  re- 
membered that  the  work  done  by  a  given  organism  depends 
upon  the  medium  and  on  the  surrounding  conditions,  or 
environment.  A  given  species  may  produce  a  typical  fer- 
mentation when  grown  in  a  medium  containing  sugar,  and, 
when  transplanted  to  albuminous  material  it  may  cause  an 
equally  typical  putrefaction.  Moreover,  the  same  organism 
may  produce  gas,  or  when  introduced  into  the  animal  body 
may  give  rise  to  disease.  The  above  terms,  therefore,  are  to 
be  taken  as  referring  to  the  most  pronounced  action  or 
function  exhibited  by  an  organism  under  certain  conditions. 


Fermentations. 


The  change  brought  about  by  an  organized  ferment  (bac- 
teria, yeasts,  moulds,  etc.),  whereby  complex  organic  bodies 
are  broken  up  into  relatively  simpler  compounds,  may  be 
designated  as  a  fermentation.  It  is  evident,  therefore,  that 
fermentations  are  vital  phenomena,  the  result  of  the  activ- 
ity of  micro-organisms.  From  what  has  been  said  hereto- 
fore, it  is  clear  that  these  organisms  induce  fermentative 


"94  BACTERIOLOGY. 

changes  largely,  if  not  wholly,  by  means  of  the  soluble  fer- 
ments or  enzymes  which  they  elaborate. 

Diverse  fermentations  result,  depending  upon  the  kind 
of  organism  at  work,  and,  the  material  that  is  acted  upon. 
It  has  been  customary  to  restrict  the  term  fermentation  to 
those  phenomena  where  carbohydrates  undergo  cleavage. 
The  word  fermentation  (meaning,  to  boil)  was  originally  ap- 
plied to  those  changes  which  were  accompanied  by  the  ac- 
tive evolution  of  gas,  in  which  case  the  liquid  appeared  to 
be  boiling.  This  conception  must  be  enlarged  to  accord 
with  the  action  of  bacteria  in  general.  It  matters  not 
whether  the  substance  that  is  acted  upon  is  a  carbohydrate, 
fat,  or  protein;  whether  gaseous  products  are,  or  are  not 
evolved;  the  change,  if  due  to  a  micro-organism,  is  one  of 
fermentation. 

In  the  decomposition  of  albuminous  substances  dis- 
agreeable products  are  evolved.  Putrefaction  can,  conse- 
quently, be  designated  as  putrid  fermentation.  The  chem- 
ical composition  of  the  protein  molecule  accounts  for  the 
different  products  thus  obtained  as  compared  with  those 
which  result  in  the  decomposition  of  carbohydrates.  The 
latter  contain  only  C,  H  and  O,  and,  on  fermentation,  yield 
diverse  alcohols  and  acids  which  are  not  disagreeable.  On 
the  other  hand,  the  protein  molecule  usually  contains  in  ad- 
dition to  the  elements  mentioned,  N,  S  and  P.  Hence,  in 
putrefaction  various  N  compounds  form  such  as  ammonia, 
amines,  indol,  skatol,  etc.,  which  possess  a  more  or  less  of- 
fensive odor.  Similarly,  the  S  may  appear  as  hydrogen 
.sulphide,  mercaptans,  etc.,  which  are  likewise  disagreeable. 

It  is  evident  that  not  only  the  material  which  is  acted 
upon,  but  also  the  particular  species  at  work  will  influence 
the  kind  of  fermentation.  Thus,  when  glucose  is  acted 
upon  by  the  yeast,  it  is  changed  to  alcohol,  whereas  another 
organism  can  convert  it  into  lactic,  or  into  butyric  acid. 
Moreover,  it  should  be  understood  that  a  given  organism 
which  induces  a  typical  fermentation  when  grown  in  a  sugar 


THE    CHEMISTRY  OF   BACTERIA.  95 

solution,  may  produce  an  equally  typical  putrefaction  when 
allowed  to  develop  on  albuminous  matter.  The  distinction 
between  saprogenic  and  zymogenic  bacteria  is,  therefore,  not 
very  marked.  Furthermore,  well  known  pathogenic  bac- 
teria may  give  rise  to  a  typical  fermentation,  or  putrefac- 
tion according  to  the  nature  of  the  material  on  which  they, 
are  grown. 

While  it  is  customary  to  speak  of  alcohol,  acetic,  buty- 
ric, lactic  acid,  etc.,  fermentations,  it  must  not  be  supposed 
that  each  one  of  these  fermentations  is  due  to  a  single  spe- 
cific organism.  It  will  presently  be  shown  that  alcoholic 
fermentation  may  be  caused  by  a  large  number  of  species  of 
yeasts,  by  many  moulds  and  even  by  bacteria.  Similarly, 
acetic  acid  fermentation  is  brought  about  by  any  one  of  a 
large  number  of  species.  The  same  is  true  of  the  other 
common  fermentations. 

The  chemical  changes  observed  in  fermentations  may 
result  in  one  of  several  ways.  By  far  the  most  common 
change  is  that  of  hydration.  In  this  case,  the  elements  of 
water  are  introduced  into  the  molecule  which  then  breaks 
up.  This  is  seen  in  the  following  equations  which  repre- 
rent  the  inversion  of  cane-sugar,  and  the  fermentation  of 

urea. 

C12H22On  -f~  H2O  =  C,jH12O6  4-  C6H]2O6. 

Cane-sugar.  Dextrose.  Levulose. 

CO(NH2)2  +  H20  =  CO,  +  2  NH:, 

Urea. 

A  second  way  in  which  cleavage  may  result  is  by 
halving.  Thus,  in  lactic  acid  fermentation,  glucose  is  appar- 
ently changed  direct  into  two  molecules  of  lactic  acid. 

C6H1206  =  2  C2H603. 

Glucose.  Lactic  acid. 

A  third  and  very  important  change  observed  in  certain 
fermentations  is  that  of  oxidation.  The  best  illustration  of 


96  BACTERIOLOGY. 

this  type  of  fermentation  is  that  seen  in  the  conversion  of 
alcohol  into  acetic  acid. 

C2H6O  +  O2  =  C2H4O2  +  H2O. 

Alcohol.  Acetic  acid. 

The  oxidation  of  NH3  to  HNO2  and  to  HNO3  is  another 
illustration. 

Lastly,  the  fermentation  process  may  be  largely  one  of 
reduction.  Thus,  nitrates  may  be  reduced  to  nitrites  and 
even  to  ammonia.  Glycerin  may  be  changed  in  this  way 
into  ethyl  alcohol.  Reduction  changes  can  be  easily  recog- 
nized if  the  medium  has  been  previously  colored  with 
litmus.  The  medium,  in  that  case,  becomes  colorless. 

Alcoholic  fermentation. — This  fermentation  is  of  the 
utmost  practical  importance,  inasmuch  as  all  the  alcohol  of 
commerce  is  thus  obtained.  The  yeasts  are  especially 
endowed  with  the  power  of  producing  alcohol.  It  should 
be  remembered  that  this  group  of  plants  differs  from  the 
bacteria  in  many  important  respects  (p.  16,  also  Chapter 
XII).  Moreover,  there  are  a  large  number  of  species  of 
yeasts  which  differ  not  only  in  the  amount  of  alcohol  to 
which  they  can  give  rise,  but  also  in  the  amount  and  char- 
acter of  other  by-products.  It  is  the  presence  of  these 
secondary  products  which  influences  to  a  large  extent,  the 
appearance,  odor  and  taste  of  beer  and  other  alcoholic 
liquors.  In  this  fermentation,  as  it  has  been  carried  on 
since  ancient  times,  it  was  always  possible  for  diverse 
yeasts  and  bacteria  to  develop,  and  thus,  more  or  less,  alter 
the  final  product.  This  uncertainty  in  the  process  has  been 
overcome  by  the  employment  of  a  pure  culture  of  a  yeast 
of  known  power.  The  work  in  large  breweries  has  thus 
been  placed  on  a  scientific  basis.  The  pure  yeast  which  is 
used  must  be  free  from  other  species  of  yeasts  as  well  as 
from  bacteria.  In  this  way  a  constant  product  is  assured. 


THE    CHEMISTRY  OF    BACTERIA.  97 

In  the  production  of  wine,  the  yeast-plant  is  not  added  to 
the  grape-juice.  It  is  present  upon  the  surface  of  the 
grapes,  and  hence,  passes  into  the  liquid  when  the  grapes 
are  crushed.  As  yet,  pure  cultures  of  yeast  have  not  been 
employed  in  the  manufacture  of  wine. 

The  best  known  species  of  the  yeasts  is  the  saccharo-' 
myces  cerevisice.  Some  species  will  produce  more  alcohol 
than  others.  Beer,  as  a  rule,  contains  less  than  5  per  cent., 
but  in  other  sugar  solutions,  as  in  wines,  the  amount  of 
alcohol  may  rise  to  10  or  15  per  cent.  This  may  be  consid- 
ered as  the  maximum  limit  of  alcohol  formation,  inasmuch 
as  this  amount  tends  to  inhibit  the  growth  of  the  yeast. 
The  so-called  "wild-yeasts"  or  torulae  do  not  yield  more 
than  about  one  per  cent,  of  alcohol. 

The  production  of  alcohol  is  not  restricted  to  the  yeast 
family.  There  are  many  bacteria  which  can  convert 
glycerin  into  alcohol.  Friedlander's  bacillus  of  pneu- 
monia when  grown  on  sugar  media  produces  alcohol  and 
acetic  acid.  The  same  is  true  of  the  typhoid  bacillus.  A 
number  of  bacteria  which  are  present  in  the  mouth  and  in 
the  intestines  of  normal  persons  are  capable  of  producing- 
alcohol.  Many  moulds,  especially  mucors,  possess  the 
power  of  forming  alcohol  out  of  sugar,  and  at  least  one  of 
these  organisms  is  utilized  on  a  commercial  scale  for  the 
preparation  of  alcohol. 

In  alcoholic  fermentation  alcohol  is  the  chief,  the 
characteristic  product.  A  large  number  of  secondary  or 
by-products  are  always  present.  Among  these  may  be 
mentioned,  carbonic  acid,  aldehyde,  the  hig-her  alcohols 
such  as  propyl,  butyl  and  amyl,  which  are  commonly  known 
as  fusel  oil.  Glycerin,  acetic  and  succinic  acids  are  likewise 
always  present. 

The  yeast-cell  when  destroyed  by  a  process  of  grind- 
ing yields  a  soluble  ferment,  an  enzyme,  which  can  convert 
sugar  solutions  into  alcohol  and  carbonic  acid.  This  fer- 
ment is  made  and  stored  up  within  the  cell.  It  may  possi- 


98  BACTERIOLOGY. 

sibly  diffuse  into  the  surrounding-  liquid,  and  cause  there  the 
characteristic  cleavage  of  sugar  into  alcohol.  It  has  been 
usually  held  that  the  sugar  was  absorbed  by  the  cell  and 
then  split  into  alcohol  and  carbonic  acid.  While  the  yeast 
decomposes  most  of  the  sugar  into  alcohol  and  other  pro- 
ducts, it  utilizes  a  small  portion  for  the  purpose  of  building 
up  its  own  cell  contents.  Cellulose  and  glycogen  may  be 
formed  direct  from  the  sugar,  whereas  the  fats  and  proteins 
result  indirectly. 

The  yeast-plant  cannot  convert  starch,  or  cane-sugar 
directly  into  alcohol.  .These  substances  must  first  be  con- 
verted into  glucose.  The  diastase  present  in  malt  is  em- 
ployed to  convert  the  starch  of  a  grain  into  sugar.  In 
Japan  and  China  certain  moulds  which  possess  a  marked 
diastatic  action  are  employed  for  this  purpose.  On  the 
other  hand,  maltose  and  cane-sugar  are  changed  by  a 
ferment  produced  by  the  yeast-cell,  invertin,  into  glucose 
which  is  then  converted  into  alcohol  and  carbonic  acid. 

C12H22On  +  H20  -  C6H1206  +  C6H1206. 

Cane-sugar.  Dextrose.  Levulose. 

C6H1206  =  2C2H60  +  2C02. 

Invert-sugar.  Alcohol. 

As  is  well  known,  the  yeast  is  also  utilized  in  the  mak- 
ing of  dough.  It  has  been  mentioned  above  that  the  yeast 
cell  is  unable  to  act  directly  upon  starch.  It  has  been  sup- 
posed that  the  bacteria  present  invert  the  starch  and  thus 
enable  the  yeast  to  carry  on  an  alcoholic  fermentation.  As 
a  result  of  the  formation  of  carbonic  acid  the  mass  is 
distended. 

Acetic  acid  fermentation. — Alcoholic  liquors  such  as  wine, 
beer,  cider,  etc. ,  on  exposure  to  air  undergo  acetic  acid  fer- 
mentation. The  alcohol  is  oxidized  to  acetic  acid  and  the 
resulting  liquid  is  commonly  known  as  vinegar.  The  free 


THE    CHEMISTRY  OF    BACTERIA.  99 

access  of  oxygen  is  necessary  to  this  change,  which  is  repre- 
sented by  this  equation: 

C2H6O  +  O2  =  C2H4O2  +  H2O. 

Alcohol.  Acetic  acid. 

It  was  supposed  by  Liebig  that  this  was  a  purely  chem- 
ical reaction,  but  the  studies  of  Pasteur  showed  that  living 
organisms  were  necessary  to  effect  the  change.  The  liquid 
undergoing  this  kind  of  fermentation  becomes  covered  with 
a  slimy  scum,  which  in  reality  is  a  zooglea,  or  mass  of  various 
bacteria.  Inasmuch  as  experience  has  taught  that  the  con- 
version of  alcoholic  liquids  into  vinegar  was  hastened  by 
the  addition  of  some  of  this  scum,  the  latter  came  to  be 
known  as  ' '  mother  of  vinegar. "  It  was  subsequently 
named  mycoderma  aceti.  This  term,  however,  must  not  be 
understood  to  designate  a  single  bacterial  species. 

The  production  of  acetic  acid  is  not  limited  to  any  one 
organism.  From  the  mother  of  vinegar  various  kinds  of 
bacteria  have  been  isolated.  As  yet,  pure  cultures  have  not 
been  employed  for  the  preparation  of  vinegar.  Among  those 
which  have  been  studied  may  be  mentioned  the  M.  aceti, 
Bacterium  aceti,  Bacillus  aceticus,  B.  Pasteurianus,  etc. 
These  organisms  exhibit  a  marked  tendency  to  give  rise  to 
involution  forms,  especially,  when  grown  at  about  40° 
(Fig.  3,  p.  21). 

Acetic  acid  fermentation  does  not  occur  in  liquids  which 
contain  more  than  15  per  cent/ of  alcohol.  Moreover,  it  is 
not  possible  to  obtain  by  direct  fermentation  a  vinegar 
which  will  contain  more  than  about  14  per  cent,  of  acetic 
acid.  A  temperature  of  30-35°  is  most  favorable  to  the 
change.  When  the  nutrient  material  becomes  exhausted 
the  bacteria  present  may  attack  the  acetic  acid  which  they 
produced,  and,  may  oxidize  this  to  carbonic  acid  and  water. 

C2H402  +  2  0,  =  2  C02  +  2  H2O. 


100  BACTERIOLOGY. 

It  has  been  indicated  above  that  acetic  acid  fermenta- 
tion is  not  a  specific  process,  due  to  a  single  organism.  Sev- 
eral bacteria  have  already  been  mentioned  as  being  capable 
of  producing  acetic  acid.  In  these  cases  the  acid  is  made 
out  of  alcohol.  In  some  instances,  it  can  apparently  be 
made  direct  from  sugar.  This  is  true  of  the  pneumococcus. 
Moreover,  it  is  a  frequent  product  in  the  anaerobic  putre- 
faction of  .proteins.  The  common  intestinal  bacteria, 
such  as  B.  lactis  aerogenes  and  B.  coli  communis,  pro- 
duce acetic  acid  together  with  formic  and  lactic  acids 
and  other  products.  These  acids,  when  formed  in  the 
intestines  of  infants,  are  unquestionably  intense  irritants, 
and  may,  therefore,  be  considered  as  factors  in  the  pro- 
duction of  infant  diarrhea.  Consequently,  the  summer 
complaint  of  infants  is  essentially,  an  abnormal  fermenta- 
tion in  the  intestines. 

Lactic  acid  fermentation. — An  unusually  large  number  of 
bacteria  are  capable  of  producing  lactic  acid,  as  the  chief 
product,  in  the  decomposition  of  sugar  and  other  carbohy- 
drates. As  in  other  fermentations  various  products  are 
present,  though  in  small  amounts.  Carbonic  acid  is  usually 
present,  accompanied  by  traces  of  alcohol,  acetone,  formic 
and  acetic  acids. 

Owing  to  the  wide  distribution  of  lactic  acid  bac- 
teria this  type  of  fermentation  will  be  frequently  met 
with.  Thus,  in  the  souring,  of  milk,  of  sugar-beet  juice, 
in  pickles,  sauer-kraut,  ensilage,  e.tc.,  lactic  acid  is  a 
characteristic  product.  It  plays  an  important  part,  more- 
over, in  certain  affections  of  the  mouth,  stomach  and  intes- 
tines. 

A  temperature  of  30-35°  is  most  favorable  to  lactic 
acid  fermentation.  The  latter  ceases  when  about  0.75  per 
cent,  of  lactic  acid  has  formed.  The  material  usually 
acted  upon  is  glucose,  or  lactose,  but  lactic  acid  may  be 
formed  from  various  other  compounds.  The  transformation 


THE    CHEMISTRY  OF    BACTERIA.  101 

of  glucose  into  lactic  :acid  may  be  expressed  by  the 
equation: 

C6H1206  =  2  C3H603. 

This  equation  is  not  strictly  correct,  inasmuch  as  small 
amounts  of  by-products  are  always  present.  Sometimes 
considerable  amounts  of  carbonic  acid  and  of  hydrogen 
may  form. 

The  lactic  acid  which  usually  forms  in  this  kind  of 
fermentation  is  optically  inactive.  It  does  not  rotate, 
either  to  the  right  or  left,  a  ray  of  polarized  light.  Some 
organisms,  however,  give  rise  to  sarco-  or  para-lactic  acid 
whereas  others  form  levo-rotatory  lactic  acid.  The  union 
of  the  latter  with  sarco-lactic  acid,  which  is  dextro-rotary, 
results  in  the  formation  of  the  ordinary  inactive  lactic  acid. 
It  is  interesting  to  note  in  this  connection  that  under  cer- 
ditions  the  colon  bacillus,  which  ferments  lactose  as  well 
as  glucose,  produces  dextro-lactic  acid,  whereas  the  typhoid 
bacillus  ferments  glucose  but  not  lactose,  and  gives  rise  to 
levo- lactic  acid.  Depending  upon  the  kind  of  sugar  acted 
upon,  the  colon  bacillus  may  give  rise  to  levo-,  dextro-,  or 
inactive  lactic  acid. 

In  dental  caries,  lactic  acid  fermentation  is  ascribed  an 
important  role.  The  organisms  present  in  the  mouth  are 
supposed  by  Miller,  to  elaborate  lactic  acid  out  of  the 
remnants  of  starchy  food,  left  between  the  teeth  or  at  the 
edge  of  the  gums.  This  acid  then  unites  with  the  calcium 
of  the  teeth  and  thus  causes  decalcification.  This  first 
stage  is  followed  by  a  second,  wherein  the  organic  matter 
of  the  tooth  is  softened  and  dissolved,  by  the  peptonizing 
bacteria  that  are  present. 

In  abnormal  fermentations  in  the  stomach  (dyspepsia) 
lactic  acid  is  a  common  product.  These  decompositions 
are  apt  to  occur  when  the  amount  of  free  hydrochloric  acid 
is  diminished,  or  when  the  food  remains  too  long  in 


102 


BACTERIOLOGY. 


the  stomach.  In  some  instances,  acetic,  or  butyric  acid 
may  predominate  in  the  stomach  contents,  and,  at  other 
times  considerable  gas  may  be  given  off.  The  production 
of  lactic  and  acetic  acids  in  the  intestines  has  already  been 
referred  to,  in  connection  with  infant  diarrhea. 

The  most  common  illustration  of  lactic  acid  fermenta- 
tion is  met  with  in  the  souring-  of  milk.  This  secretion,  as 
elaborated  in  the  gland,  is  strictly  sterile,  and  if  col- 
lected under  suitable  precautions  it  will  keep  indefinitely. 
In  ordinary  milking-  an  enormous  number  of  bacteria  are 
introduced  into  the  milk,  from  the  air  and  through  unclean 
hands,  containers,  etc.  Given  a  suitable  temperature,  these 
org-anisms  rapidly  multiply  and  convert  the  milk-sugar  into 
lactic  acid.  As  a  result,  the  reaction  of  the  milk,  which  was 
neutral  or  alkaline  in  the  beginning,  becomes  acid.  Inas- 
much as  the  casein  is  insoluble  in  the  presence  of  an  acid, 
it  forms  a  precipitate  which  mechanically  drags  down  the 
fat.  The  curdling  of  milk  may  result  from  the  action  of  a 
rennet  ferment,  but,  as  a  rule,  it  is  due  directly  to  the  acid 
reaction  which  develops  in  the  milk. 

It  is  evident  that  some  bacteria  will  coagulate  milk, 
whereas  others  will  not.  This  fact  is  made  use  of,  at  times, 
in  differentiating  bacteria.  Thus,  milk  is  coagulated  by 
the  colon  bacillus  but  is  not  altered  by  the  typhoid  bacillus. 

In  eastern  Europe  and  in  Asia,  the  milk  of  the  cow,  ass, 
or  camel  is  subjected  to  an  alcoholic,  as  well  as  lactic 
acid  fermentation.  The  resultant  beverage  is  known  as 
JcepJiyr  or  koumiss.  The  fermentation  is  induced  by  adding 
to  the  milk  the  so-called  kephir  grains,  which  consist 
essentially  of  bacteria  and  yeasts. 

The  lactic  acid  bacteria  favor  the  development,  in  the 
body,  of  anaerobic  bacteria,  such  as  the  tetanus  bacillus. 
Microbic  associations  of  this  kind  play  an  important  part 
in  many  diseases. 

It  may  be  well  to  briefly  allude  in  this  connection  to  the 
so-called  ripening  of  cheese.  Bacteria  are  to  a  very  large 


THE    CHEMISTRY  OF    BACTERIA.  103 

extent,  if  not  wholly,  the  cause  of  this  change.  As  a  result 
of  gas  production,  vacuoles  or  spaces  form  in  the  mass. 
The  characteristic  flavor  and  odor  is  due,  as  in  the  case  of 
butter,  to  bacterial  products.  Abnormal  fermentations  in 
a  cheese  may  give  rise  to  poisonous  products,  or  to  marked 
alteration  in  the  appearance  of  the  cheese. 

Butyric  acid  fermentation. — This  rather  common  fermen- 
tation is  usually  the  result  of  the  action  of  anaerobic  bac- 
teria. A  considerable  number  of  obligative  anaerobes  have 
been  described  as  producers  of  butyric  acid.  The  patho- 
genic organisms  of  this  class  may  form  butyric  acid,  even  in 
the  animal  body.  A  number  of  aerobic  bacteria  can  like- 
wise give  rise  to  butyric  acid. 

Many  of  the  anaerobes  mentioned,  give  a  granulose  re- 
action on  contact  with  iodine.  They  are  usually  motile, 
and  in  those  species  which  develop  spores,  there  is  always 
a  corresponding  enlargement  of  the  cell.  Hence,  either  the 
clostridium,  or  drum-stick  forms  are  met  with  in  such  cases. 

In  this,  as  in  other  fermentations,  various  additional 
products  are  encountered.  Among  these  may  be  mentioned, 
butyl  alcohol,  acetic  and  carbonic  acids,  hydrogen,  etc. 
The  material  acted  upon  may  be  a  carbohydrate,  such  as 
glucose,  or  it  may  be  a  protein,  or  a  fat.  It  is  evident  that 
butyric,  like  lactic  acid,  is  a  very  common  product  in  the 
decomposition  of  organic  matter.  The  transformation  of 
glucose  into  butyric  acid  is  usually  represented  by  the 
equation: 

C6H1206  -  C4H802  +  2  C02  +  2  H2. 

Glucose.  Butyric  acid. 

Butyric  acid  is  ordinarily  prepared  by  allowing  milk,  to 
which  calcium  carbonate  has  been  added,  to  undergo  lactic 
acid  fermentation.  The  lactic  acid  unites  with  the  lime  to 
form  calcium  lactate.  Eventually,  this  salt  is  acted  upon 
by  other  organisms  and  butyric  acid  forms. 


104  BACTERIOLOGY. 

In  the  fermentation  of  cellulose,  anaerobic  bacteria  play 
a  most  important  role.  The  cellulose  is  first  converted  into 
sugar,  which  in  turn  yields  butyric  acid,  marsh-gas,  and 
diverse  other  products.  The  changes  that  take  place  in  the 
production  of  sauer-kraut,  ensilage,  etc. ,  are  similar  to  that 
just  [mentioned.  The  cellulose  is  softened,  and  partially 
fermented  with  the  production  of  butyric  and  lactic  acids. 
In  the  ripening  of  cheese,  similar  decomposition  products 
are  formed  out  of  the  casein.  The  retting  of  hemp  and  flax 
is  a  change  analogous  to  the  fermentation  of  cellulose.  The 
fibres  are  held  together  by  a  cementing  substance,  calcium 
pectate,  which  is  removed  by  allowing  the  material  to  soak 
in  water.  The  intercellular  material  is  not  dissolved  by  the 
water,  but  is  digested,  or  dissolved  by  the  action  of  butyric 
acid  bacteria. 

Viscous  or  slimy  fermentation. — It  not  infrequently  hap- 
pens that  milk,  urine,  beer,  wine  and  dilute  sugar  solutions 
take  on  a  slimy  or  ropy  consistence.  This  is  due  to  the 
presence  and  development  of  certain  bacteria.  As  might 
be  expected  a  very  large  number  of  wholly  different  organ- 
isms are  capable  of  imparting  a  slimy  character  to  these 
several  liquids.  It  may  happen  that  such  bacteria  will  de- 
velop in  sausage,  bread,  or  in  biscuits.  In  such  cases,  on 
breaking  some  of  the  material  and  slowly  drawing  the 
pieces  apart,  they  will  be  seen  to  be  connected  by  numerous, 
fine,  cobweb-like  threads.  The  slimy  covering  of  the  ton- 
gue, teeth,  etc.,  in  many  fevers,  is  probably  due  to  organ- 
isms of  this  kind. 

The  slimy  character  may  be  due  to  one  of  two  causes. 
In  the  first  place,  the  organism  may  secrete,  or  throw  off  a 
slimy  product.  This  material  passes  out  into  the  surround- 
ing liquid  and  is  no  longer  connected  with  the  cell.  This 
condition  is  analogous  to  the  secretion  of  mucin  by  the  cells 
of  the  mucous  membrane.  On  the  other  hand,  the  sliminess 
may  be  due  to  the  formation  of  capsules.  It  has  been 


THE    CHEMISTRY  OF    BACTERIA.  105 

pointed  out  (p.  28)  that  the  cell- wall  of  many  bacteria  may 
soften  or  gelatinize,  giving-  rise  to  the  so-called  capsule. 
The  organisms,  in  such  cases,  tend  to  stick  together,  form- 
ing' zoogleae. 

Several  of  these  fermentations  are  of  interest,  because 
the  gummy  substance  which  is  produced,  known  as  visa 
or  dextran,  is  a  carbohydrate  allied  to  dextrin.  Its  formula, 
C6H10O5  is  the  same  as  that  of  dextrin.  Among-  the  by-pro- 
ducts in  this  case  may  be  mentioned  carbonic  and  lactic 
acids,  and  mannite.  The  latter  probably,  results  by  the  ac- 
tion of  nascent  hydrogen  on  glucose.  The  Leuconostoc  mes- 
enterioides  is  a  common  cause  of  such  fermentations  in  molas- 
ses, etc.,  in  sugar  refineries.  This  organism,  when  grown 
in  sugar  solutions,  develops  enormous  capsules,  which  are 
10  or  20  times  the  width  of  the  cell  itself.  On  the  usual 
nutrient  media  it  appears  as  an  ordinary  streptococcus. 
Under  favorable  conditions  it  may  develop  with  extreme 
rapidity,  converting  the  sugar  solution  into  a  jelly,  or  even 
into  a  cartilaginous  mass.  In  one  instance,  80  barrels  of 
molasses  became  converted  into  a  gelatinous  mass  within 
12  hours  A  temperature  .of  30-35°  favors  this  fermentation. 

Fermentation  of  fats. — The  various  animal  and  vegetable 
fats  or  oils  are  essentially  neutral  compounds  of  glycerin 
and  one  or  more  fatty  acids.  A  strictly  pure  fat  cannot  be 
acted  upon  by  bacteria,  inasmuch  as  it  contains  only  C,  H 
and  O.  It  may,  however,  become  rancid  by  exposure  to  the 
action  of  air  and  light.  The  change  that  results  consists 
in  the  splitting  of  the  fat  into  its  components,  glycerin  and 
fatty  acids.  If,  on  the  other  hand,  the  fat  is  not  pure  but 
contains  nitrogenous  and  other  matter  it  will  be  possible 
for  bacteria  to  grow  in  .such  a  mass.  In  that  event,  a  more 
or  less  extensive  fermentation,  or  cleavage  of  the  fat  will 
occur. 

The  decomposition  of  fat  by  means  of  bacteria  is  a 
constant  occurrence  in  the  intestines.  The  change  which 


106  BACTERIOLOGY. 

these  organisms  induce  is  analogous  to  that  produced  by 
the  fat-splitting  ferment  of  the  pancreatic  juice. 

Butter,  as  is  well  known,  is  apt  to  become  rancid, 
especially,  if  it  has  been  imperfectly  worked.  In  that 
case,  more  or  less  casein  and  other  milk  constituents 
remain  in  the  mass  of  butter-fat  and  furnish  the  neces- 
sary soil  for  the  development  of  bacteria.  Inasmuch  as 
butter  contains,  among  other  fats,  a  small  amount  of  a 
butyrate,  the  latter  on  decomposition  will,  of  course, 
yield  butyric  acid.  The  odor  of  strong  butter  is  largely 
due  to  this  acid.  It  may  be  well  in  this  connection  to 
mention  that  the  aroma  of  a  good  butter  is  largely  due  to 
volatile  ethereal  products  made  by  bacteria.  Certain 
organisms,  which  possess  the  power  of  improving  the 
flavor,  when  planted  in  the  cream  will  render  an  excellent 
butter  out  of  what  otherwise  might  have  been  a  poor  grade 
article. 

Fatty  acids  are  not  only  liberated  in  the  decomposition 
of  fats,  but  they  may  also  form  in  the  cleavage  of  proteins. 
The  fatty  acids  in  the  free  state  are  not  readily  split  up 
into  simpler  products  by  bacteria.  When  they  unite  with 
metals  to  form  salts  they  are  more  prone  to  fermentation. 
In  that  case  they  are  oxidized  and  form  carbonic  acid  and 
other  compounds.  Thus,  calcium  formate  yields  calcium 
carbonate,  carbonic  acid  and  hydrogen,  whereas  calcium 
acetate  will  give  rise  to  the  same  products  except  that  the 
hydrogen  is  replaced  by  marsh-gas.  The  calcium  salts  of 
the  higher  fatty  acids  are  split  up  into  a  variety  of  pro- 
ducts. The  glycerin,  likewise,  undergoes  fermentation, 
forming  butyric,  lactic  and  carbonic  acids. 

Hydrogen  sulphide  fermentation. — In  many  instances, 
when  certain  sulphur  compounds  are  present  in  a  ferment- 
ing liquid  they  are  converted  into  hydrogen  sulphide.  In 
minute  quantity,  this  gas  is  formed  by  nearly  all  of  the 
pathogenic  bacteria,  and,  inasmuch  as  it  is  a  highly  poison- 


THE    CHEMISTRY  OF    BACTERIA.  107 

ous  substance,  it  may  not  be  without  effect  when  formed  in 
the  tissues,  in  disease  conditions. 

An  interesting-  fermentation  of  this  type  is  occasion- 
ally met  with  in  urine.  Bacteria,  when  introduced  into  the 
bladder,  either  as  the  result  of  an  injury,  or  by  the  use  of 
instruments  or  otherwise,  may  develop  and  give  rise  to  fer- 
mentative •  changes.  Usually  an  ammoniacal  decomposi- 
tion results,  but  at  times  the  urine  remains  strongly  acid 
.and  possesses  a  marked  odor  of  hydrogen  sulphide.  This 
condition,  or  hydrogen  sulphide  fermentation  of  the  urine,  is 
known  as  hydrothionuria.  A  number  of  bacterial  species 
have  been  isolated  by  the  author  and  by  others  from  such 
urines,  and  when  inoculated  into  sterile,  normal  urine  they 
promptly  produce  this  gas.  The  so-called  ''neutral"  sul- 
phur compounds  of  the  urine,  and  not  the  sulphates,  are 
acted  upon.  Hydrogen  sulphide  is  a  common  product,  in 
the  decomposition  of  protein  substances.  The.  odor  of 
rotten  eggs  is  due  to  this  gas. 

Ammoniacal  fermentation  of  urine. — Normal  urine  is  per- 
fectly free  from  bacteria  for  reasons  heretofore  given  (p.  65). 
When  bacteria  are  introduced  into  the  bladder,  in  some  way 
as  mentioned  above,  they  usually  induce  a  marked  ammon- 
iacal decomposition.  The  urea  undergoes  hydration,  form- 
ing ammonia  and  carbonic  acid,  which,  of  course,  unite  to 
form  ammonium  carbonate.  The  same  fermentation  will 
almost  invariably  occur  when  normal  urine  is  allowed  to 
stand  for  some  hours  at  a  warm  temperature.  The  change 
corresponds  to  the  equation: 

CO(NH2)2  +  H2O  =  CO2  +  2  NH8. 

Urea. 

Pasteur  designated  the  cause  of  this  fermentation  as 
the  Micrococcus  urece.  The  change,  however,  is  not  spe- 
cific, due  to  a  single  organism,  but  can  be  induced  by  a 


108  BACTERIOLOGY. 

host  of  aerobic  as  well  as  anaerobic  bacteria.     These  are 
found  everywhere  in  nature,  in  the  air,  water  and  soil. 

Ammonia,  like  hydrogen  sulphide,  is  a  very  common 
product  formed  in  the  putrefaction  of  protein  substances, 
whether  of  animal  or  vegetable  origin.  Diverse  bacteria 
and  moulds  take  part  in  this  transformation. 

Nitrification. — As  indicated  above,  the  albuminous  sub- 
stances present  in  the  dead  plant  or  animal,  are  acted  upon 
by  bacteria  and  decomposed  into  the  simplest  inorganic 
products.  The  nitrogen  eventually  appears  as  ammonia, 
although  a  small  amount  may  be  returned  to  the  air  as  the 
free  element.  The  ammonia,  thus  formed,  is  essential  to 
the  growth  of  higher  plants.  In  order  to  be  assimilated  it 
must  be  oxidized.  This  change  is  again  effected  by  certain 
bacteria,  which  oxidize  the  ammonia  first  to  nitrous,  and 
finally  to  nitric  acid. 

Organisms  of  this  kind  are  widely  distributed  in  the 
soil  and  in  water.  Many  of  the  ordinary,  pathogenic  and  non- 
pathogenic  bacteria,  are  capable  of  forming  nitrites  and  ni- 
trates. This  change  can  be  readily  observed  in  urine,  which 
ordinarily  contains  only  traces  of  these  compounds.  As 
soon  as  bacteria  begin  to  develop  in  the  urine,  nitrites  and 
nitrates  can  be  detected.  It  is  for  this  reason  that  the 
chemist,  when  examining  water  suspected  of  being  polluted, 
tests  for  nitrites  and  nitrates,  as  well  as  for  ammonia. 

The  formation  of  nitrites  and  nitrates  is,  therefore,  tak- 
ing place  wherever  dead,  nitrogenous,  plant  or  animal  mat- 
ter is  present.  The  water,  and  especially  the  soil,  contains, 
bacteria  which  are  capable  of  effecting  this  conversion.  A 
suitable  temperature,  moisture,  and  a  proper  amount  of 
oxygen  are,  of  course,  likewise  essential.  It  is  in  this  way  that 
the  dung-heaps  of  India  furnished  the  world's  supply  of  salt- 
peter. The  soda  salt-peter  which  exists  in  vast  deposits  in 
Chili  and  Peru,  likewise,  owes  its  origin  to  the  action  of 
bacteria  on  the  guano,  or  excrement  of  birds. 


THE    CHEMISTRY  OF   BACTERIA.  109 

It  may  be  of  interest  to  note  that  nitrites  are  almost  al- 
ways present  in  the  saliva,  as  a  result  of  the  action  of  the 
mouth  bacteria  upon  the  food  constituents.  Nitrates  do  not 
seem  to  be  present. 

Among  the  nitrifying  organisms  there  are  some  which 
differ  markedly  from  all  other  known  bacteria.  Unlike 
latter,  they  do  not  require  organic  matter  inasmuch  as  they 
are  able  to  assimilate  carbonic  acid.  Without  the  aid  of 
chlorophyll,  and  without  light,  they  can  utilize  the  carbonic 
of  the  air  as  food.  Not  only  is  organic  matter  unnecessary, 
but  it  is  even  injurious,  and  retards  the  growth  of  these 
bacteria.  Hence,  special  methods,  wholly  unlike  those  or- 
dinarily employed  when  cultivating  bacteria,  must  be  re- 
sorted to,  in  order  to  isolate  these  remarkable  organisms. 
They  can  be  grown  best  in  pure  water  to  which  0. 1  per  cent, 
of  ammonium  sulphate  and  of  potassium  phosphate,  and 
some  basic  magnesium  carbonate  has  been  added.  They 
can  also  be  isolated  by  growing  upon  a  nitrite-agar,  or  upon 
a  mineral-gelatin  which  is  essentially  silicic  acid. 

In  view  of  the  cultural  requirements  of  these  organisms 
it  will  be  seen  that  they  belong  to  the  simplest  and  earliest 
forms  of  life.  They  exist  on  wholly  inorganic  matter,  since 
the  carbonic  acid  of  the  air  supplies  the  necessary  carbon, 
while  ammonia  yields  the  nitrogen  essential  to  the  forma- 
tion of  protoplasm.  These  organisms  convert  the  ammonia, 
by  process  of  oxidation,  first  to  nitrous  acid  and  then  to 
nitric  acid.  These  changes  can  be  indicated  by  the  equa- 
tions: 

2  NH3  +  •  3  Oa  =  2  HNO2  4-  2  H2O. 
2  HNO2  +  O2  =  2  HNO3. 

While  there  are  nitrifying  organisms  which  can  change 
ammonia  into  nitrates,  there  are  many  others  which  can 
produce  nitrites,  but  not  nitrates.  Other  organisms,  in  that 
case,  complete  the  process,  converting  the  former  com- 


110  BACTERIOLOGY. 

pounds  into  the  latter.  Hence,  the  nitrite-forming  or  ni- 
troso-bacteria  must  be  supplied  with  an  ammonium  salt, 
whereas  the  nitrate-producing'  forms,  or  nitro-bacteria  re- 
quire a  salt  of  nitrous  acid. 

The  formation  of  nitrites  and  of  nitrates  in  the  soil  is, 
therefore,  as  Pasteur  pointed  out  in  1862,  a  process  analog- 
ous to  acetification.  The  production  of  acetic  acid  and  of 
nitric  acid  is  due  to  oxidation  changes,  which  are  not  purely 
chemical,  as  was  at  one  time  supposed.  These  changes  are 
dependent  upon  the  presence  of  certain  organisms.  Nitri- 
fication is,  therefore,  a  fermentation  analogous  to  that  of 
acetic  acid.  It  is  not  one  organism  but  rather  a  large 
number,  or  group  of  organisms,  which  possess  the  power  of 
inducing  these  fermentative  changes. 

A  striking  contrast  to  the  action  of  these  organisms  is 
that  of  the  de-nitrifying  bacteria.  The  latter  possess 
remarkable  reducing  powers.  They  convert  the  nitrates 
into  nitrites,  and  continue  the  reduction  until  all,  or  nearly 
all,  of  the  nitrogen  has  been  set  free,  as  such.  This  appar- 
ent waste  of  nitrogen  may  be  considered  as  being  balanced 
by  the  activity  of  the  bacteria  which  are  present  in  the  root 
nodules  of  leguminous  plants.  These  organisms  are  able 
to  assimilate,  or  fix  the  atmospheric  nitrogen,  and  trans- 
fer it  to  their  host.  There  are,  therefore,  four  groups  of 
bacteria  according  to  their  action  on  nitrogen  and  its 
inorganic  compounds. 

1. — Those  which  cause  a  fixation  of  free  nitrogen. 
2. — Those  which  produce  nitrites  out  of  ammonia. 
3. — Those  which  produce  nitrates  out  of  nitrites. 
4. — Those  which  reduce  nitrates  to  nitrites  and  to  free 
nitrogen. 

A  further  illustration  of  the  reducing  action  of  bac- 
teria is  seen  in  the  manufacture  of  indigo.  The  indigo  does 
not  exist,  as  such,  in  the  plant,  but  rather  as  a  glucoside. 


THE    CHEMISTRY  OF   BACTERIA.  Ill 

The  cut  plants  are  placed  in  water  and  allowed  to  ferment, 
whereby  the  glucoside  is  split  up  into  its  constituents, 
sugar  and  reduced  or  white  indigo.  The  latter  passes  into 
solution,  and,  on  subsequent  agitation  with  air,  becomes 
oxidized  to  the  insoluble  indigo-blue. 

Putrefaction. 


The  decomposition  of  albuminous  substances  is  a  fer- 
mentative change  analogous  in  every  respect  to  the  changes 
which  carbohydrates  and  fats  undergo.  The  cleavage  of 
dead  organic  matter  is  always  brought  about  by  the  activity 
of  micro-organisms.  A  given  organism  may  be  capable  of 
splitting  up  carbohydrates,  and  thus,  inducing  a  typical 
fermentation  when  planted  on  a  soil  rich  in  these  com- 
pounds. The  same  germ,  when  grown  on  albuminous  matter, 
may  produce  a  typical  putrefaction.  The  difference  be- 
tween fermentation  and  putrefaction  is,  therefore,  princi- 
pally due  to  the  different  chemical  composition  of  the 
material  that  is  acted  upon. 

While  the  carbohydrates  and  fats  contain  merely 
C,  H  and  O,  the  protein  substances  contain,  in  addition, 
N,  S  and  at  times  P.  Consequently,  cleavage  products 
containing  the  latter  elements  will  be  met  with  in  putrefac- 
tion, which  can  not  be  present  in  ordinary  fermentation. 
These  products  may  possess  a  more  or  less  intensely  disa- 
greeable odor,  and  hence,  putrefaction  may  be  spoken  of  as 
putrid  fermentation.  Among  the  compounds  thus  formed  may 
be  mentioned  ammonia,  and  the  amines,  which  possess  a  fish- 
like  odor;  also,  indol  and  skatol  which  are  characteristic 
products  of  intestinal  putrefaction  and  the  cause  of  the 
fecal  odor.  These  substances  contain  the  nitrogen  which 
was  present  in  the  protein  molecule.  The  sulphur  of  the 
protein  molecule  may  appear  as  hydrogen  sulphide,  mer- 
captans,  etc. 


1 1 2  BACTERIOLOGY. 

In  the  fermentation  of  carbohydrates,  or  of  fats,  as  might 
be  expected,  various  organic  acids  form.  The  reaction  of 
the  fermenting-  liquid  is,  therefore,  acid  and  the  odor  may 
be  considered  as  pleasant.  On  the  other  hand,  in  the  fer- 
mentation of  proteins  the  ammonia  and  amines  that  form 
may  impart  an  alkaline  reaction  to  the  medium,  and,  as 
indicated  above,  disagreeable  odors  are  usually  present. 

Without  the  agency  of  micro-organisms  the  dead  ani- 
mals and  plants  would  accumulate  upon  the  earth's  surface. 
The  action  of  atmospheric  oxygen,  and  of  light,  would  be 
wholly  insufficient  to  accomplish  the  change  of  "dust  to 
dust."  The  removal  of  dead  matter  may  be  considered  as 
the  special  function  of  microscopic  life.  In  this  respect, 
the  bacteria  are  to  be  looked  upon  as  performing  a  most 
important  part — that  of  nature's  scavengers.  The  waste- 
matter  of  the  living,  and  the  lifeless  remains  themselves  are 
in  a  short  time  converted  into  the  simplest  inorganic  chem- 
ical compounds.  The  carbon  present  in  the  starch,  cellu- 
lose, or  protein  molecule  is  returned  by  the  agency  of  these 
organisms  to  the  inorganic  world  as  carbonic  acid.  In  like 
manner  the  hydrogen  becomes  converted  into  water.  The 
nitrogen  present  in  the  cell  protoplasm,  which  is  by  far  the 
most  complex  chemical  substance  known,  is  brought  back 
to  its  simplest  form  ammonia,  nitrous,  and  nitric  acids. 

Bacteria  are,  unquestionably,  the  most  active  agents  in 
this  resolution  of  dead  matter.  Other  forms  of  life,  how- 
ever, may  also  induce  similar  changes.  The  moulds  and 
yeasts,  and  even  the  infusoria,  are  deserving  of  mention  in 
this  connection. 

The  removal  of  dead  matter,  as  indicated  above,  has  one 
important  consequence  and  that  is  the  perpetuation  of  life. 
The  ordinary  plants  derive  their  food  from  the  carbonic 
acid  of  the  air  and  from  the  nitrates  of  the  soil.  The  starch, 
sugar,  fats  and  protein  matter  which  they  elaborate,  in  turn, 


THE    CHEMISTRY  OF    BACTERIA.  113 

serve  as  the  sole  food  for  the  herbivorous  animal.  The 
carnivorous  animal  derives  its  food  from  the  latter,  and 
man,  as  an  omnivorous  being,  is  consequently,  directly  or 
indirectly,  dependant  upon  the  vegetable  kingdom  for  food. 
The  nitrogen  which  as  ammonia,  nitrites  and  nitrates  was 
present  in  the  soil,  and  the  carbon,  which  as  carbonic  acid- 
was  present  in  the  air,  have  been  built  up  into  the  complex 
protoplasm  of  the  animal  cell.  On  the  death  of  the  plant 
or  animal,  these  elements  would  remain  indefinitely  in  this 
combination  were  it  not  for  the  action  of  micro-organisms. 
These  induce  the  decay  or  putrefaction,  which  results  in  the 
complete  cleavage  of  these  complex  chemical  substances, 
so  that  the  elements  are  transformed  into  the  simple  com- 
pounds in  which  they  originally  existed,  in  the  soil  and  air. 

The  carbonic  acid,  which  is  thus  liberated  in  the  fer- 
mentation or  putrefaction  of  dead  matter,  is  utilizable  by  a 
growing  plant.  As  long  as  the  carbon  was  present  in  the 
cellulose,  starch,  fat  or  protein  molecule  it  was  as  useless  to 
new  plant  life  as  that  contained  in  the  limestone  deposits 
of  the  earth.  The  same  is  equally  true  of  the  nitrogen 
stored  up  in  the  protein  molecule.  It  is  only  when  brought 
back  to  the  inorganic  form  that  it  can  serve  as  plant  food. 
The  microscopic  workers,  therefore,  in  their  r61e  of  scaven- 
gers prevent  the  accumulation  of  dead  animal  and  plant 
matter,  and,  at  the  same  time,  maintain  a  cycle  of  life  in 
which  they  themselves  form  an  important  link,  and  thus 
justify  the  axiom  "Death  is  Life." 

The  bacteria  which  produce  putrefaction  have  been  de- 
signated as  saprogenic.  These  same  organisms,  however, 
when  grown  on  media  containing  sugar  or  other  carbohy- 
drates may  give  rise  to  fermentations,  so  that  in  the  latter 
case  they  would  also  be  designated  as  zymogenic.  It  is 
evident,  therefore,  that  putrefaction  is  not  a  specific  change 
due  to  a  single  organism,  or  even  to  a  special  group  of 
organisms.  It  is  induced  by  any  one  of  a  large  number  of 
bacteria,  whenever  these  are  grown  in  solutions  of  protein 

8 


114  BACTERIOLOGY. 

substances.  As  might  be  expected,  the  products  will  differ 
according  to  the  kind  of  organism  at  work,  the  temperature 
and  the  amount  of  oxygen,  etc.  The  bacterium  termo  of 
the  older  writers,  is  to  be  considered  as  a  group  name 
covering  a  number  of  saprogenic  bacteria,  rather  than  any 
one  individual  organism. 

Pigment  Production. 

The  various  bacteria  which  give  rise  to  pigments  are 
said  to  be  chromogenic.  The  activity  of  these  organisms  is 
not  limited  to  the  production  of  pigment.  Thus,  the  well- 
known  golden,  pus-producing  micrococcus  not  only  produces 
a  pigment,  but  is  also  pathogenic,  and  may,  indeed,  induce 
typical  fermentations.  It  is  evident  that  the  pigment  is  to 
be  considered  as  one  of  the  many  chemical  products  elabor 
ated  by  the  bacterial  cell. 

The  number  of  chromogenic  bacteria  probably  exceeds 
one  hundred.  All  shades  of  color  may  be  found  among  the 
different  individuals  of  this  group.  It  has  been  shown,  here- 
tofore, that  the  majority  of  bacteria  are  perfectly  colorless. 
In  only  a  very  small  number  of  bacteria  do  the  cells  contain 
a  pigment.  The  contents  of  the  cell,  in  such  cases,  may  be 
colored  a  light  pink  or  purple,  green,  yellow  or  brown.  In 
the  first  case  the  pigment,  known  as  bacterio-purpurin, 
may  be  considered  as  allied  to  chlorophyll,  inasmuch  as  the 
organisms  which  contain  it,  unquestionably,  give  off  oxygen 
in  the  presence  of  light.  The  green  pigment,  present  in  a 
few  species  may,  in  like  manner,  be  possibly  related  to 
chlorophyll. 

With  the  exception  of  the  instances  just  mentioned,  the 
cells  of  the  chromogenic  bacteria  are  perfectly  colorless. 
It  is  clear  that  in  these  cases,  the  pigment,  or  its  antece- 
dent, is  made  within  the  cell,  and  is  excreted  or  passed  out 
as  rapidly  as  it  is  made.  Moreover,  there  is  reason  to  be- 


li^ 


THE    CHEMISTRY   OF    BACTERIA.  115 


I 


lieve  that,  in  most  of  these  cases,  the  pigment  is  not  a  prim- 
ary, bat  rather  a  secondary  product.  That  is  to  say,  the 
bacterial  cell  elaborates  a  colorless,  or  leuco-product  which, 
on  contact  with  the  oxygen  of  the  air,  becomes  converted 
into  the  pigment.  Almost  invariably,  pigment  production 
will  be  observed  on  the  surface  of  the  culture  media,  where- 
there  is  free  access  of  oxygen. 

In  a  few  instances,  as  in  the  case  of  the  Spirillum  ru- 
brum,  the  pigment  is  formed  in  the  absence  of  air.  This 
would  indicate  that  in  the  particular  case,  the  pigment  was 
a  primary  product,  made  directly  by  the  cell. 

The  pigment  production  in  a  given  species  is  subject  to 
considerable  variation.  At  one  time  the  growth  may  pos- 
sess a  very  bright  color,  and  again,  at  another  time  it  may 
be  very  pale  or  colorless.  The  chromogenic  property  is, 
therefore,  easily  influenced  by  even  trivial  conditions.  Un- 
der no  condition,  however,  does  a  given  organism  give  rise 
to  different  pigments,  although,  in  a  few  instances,  several 
pigments  may  be  formed  at  the  same  time.  The  temperature, 
reaction  and  composition  of  the  medium,  and  the  oxygen 
supply  are  factors  especially  deserving  attention. 

As  a  rule,  pigment  production  is  diminished,  or  sup- 
pressed, when  the  cultures  are  grown  at  about  the  body  tem- 
perature, 37-39°.  The  most  brilliant  pigments  are  obtained 
when  the  cultures  are  grown  at  about  15°.  Prolonged  cul- 
tivation, at  a  high  temperature,  may  result  in  the  produc- 
tion of  permanent,  colorless  varieties. 

Exposure  to  sun-light  has  a  similar  effect  as  a  high 
temperature.  In  many  instances,  an  insolation  of  a  few 
hours  may  give  rise  to  colorless  varieties. 

The  reaction  of  the  medium  has  a  considerable  influence 
upon  pigment  production.  This  will  especially  be  pointed 
out  in  connection  with  the  bacillus  of  blue  milk.  As  to  the 
composition  of  the  medium,  it  should  be  said  that  a  starchy 
surface,  such  as  that  of  a  potato,  seems  to  be  most  favor- 
able to  pigment  production. 


1 1 6  BACTERIOLOGY. 

After  the  lapse  of  a  few  days,  the  brightest  pigment 
fades,  and  may  even  disappear.  This  alteration  may  be  due 
to  the  action  of  the  air,  although  the  bacterial  products, 
especially  the  enzymes,  may  assist  in  the  change. 

Although  the  bacterial  pigments  have  been  the  subject 
of  numerous  investigations,  their  exact  chemical  nature  is 
still  unsettled.  In  some  instances,  the  pigment  would  seem 
to  be  a  fatty  compound  (lipochrome)\  in  another  instance,  it 
would  appear  to  be  a  basic  substance,  allied  to  the  ptomains 
(pyocyanin)-,  and  again,  in  other  cases,  it  appears  to  be  a 
protein  compound. 

The  various  pigments  may  be  divided  into  two  large 
groups  according  to  their  solubility  in  water.  The  fluores- 
cing  bacteria,  and  the  bacillus  of  blue  milk  afford  the  best 
examples  of  soluble  pigments.  These  pigments  form  on  the 
surface  of  the  medium,  where  there  is  an  abundance  of 
oxygen,  and  gradually  diffuse  downward  into  the  medium. 
In  the  other  group  the  pigment  is  insoluble  in  water,  and 
soluble  in  alcohol.  The  Bacillus  prodigiosus,  violaceus,  etc. , 
are  good  examples  of  this  kind.  In  these  instances  the 
pigment  exists  as  granules  on  the  outside  of  the  cells.  In 
only  two  organisms  does  the  pigment  seem  to  be  insoluble 
in  water  and  in  alcohol. 

It  is  possible  for  various  articles  of  food  such  as  meat, 
milk,  cheese,  bread,  etc.,  to  develop  growths  of  pigment 
producing  bacteria.  The  B.  prodigiosus  and  allied  forms 
have  been  frequently  met  with  in  such  "food  epidemics  " 
The  miracle  of  the  "bleeding  host,"  as  well  as  "bloody 
milk,"  imputed  in  a  former  age  to  witch-craft,  find  an  easy 
explanation  in  the  sudden  development  of  chromogenic 
bacteria. 

Phosphorescence. 

The  extremely  interesting,  and  rather  mystical  pheno- 
menon of  phosphorescence  is  common  to  the  sea-water  in 
all  parts  of  the  globe.  Salt-water  fish  and  oysters,  when 


THE    CHEMISTRY  OF    BACTERIA.  117 

kept  in  a  dark  place,  will  not  infrequently  phosphoresce. 
When  such  fish  are  kept  in  meat-shops,  it  may  happen  that 
veal,  pork  and  beef  will,  likewise,  develop  this  strange 
property.  This  phenomenon  is  met  with  especially  in  sea- 
coast  towns.  The  river  and  lake  waters  do  not  phosphor- 
esce, nor  do  the  fish  taken  from  such  waters.  Instances  of 
phosphorescence  are  met  with  inland,  in  the  well-known 
fox-  or  punk-fire  which  is  observed  in  the  woods  at  night, 
on  the  surface  of  old  stumps.  The  fire-fly  or  glow-worm 
affords  another  illustration. 

Phosphorescence,  like  pigment  production,  or  fermen- 
tation, is  a  vital  phenomenon.  It  has  been  known  for  a 
long  time  that  certain  protozoa  in  the  sea-water  emit  light. 
Moreover,  this  property  has  been  observed  in  some  algae 
and  the  ordinary  fox-fire  is  due,  unquestionably,  to  certain 
moulds.  It  is  only  within  comparatively  recent  times  that 
bacteria  have  been  shown  to  possess  this  strange  power. 
Pfliiger's  observation,  in  1874,  rendered  it  probable  that  cer- 
tain bacteria  were  the  cause  of  the  phosphorescence  ob- 
served on  fish  and  meat,  but  it  was  not  until  1887,  when 
Fischer  obtained  pure  cultures,  that  the  cause  was  de- 
monstrated. The  mysterious  glow  on  fish  and  meats  like 
the  pigmented  milk,  meat  or  starchy  food  is,  therefore,  due 
to  the  presence  and  growth  of  bacteria.  In  both  cases,  the 
microscope  has  revealed  the  cause  which  in  the  past  was 
but  too  often  ascribed  to  sorcery,  or  to  supernatural  powers. 

At  the  present  time  about  25  species  of  phosphorescing 
bacteria  are  known.  It  is  quite  likely  that  some  of  these 
may  be  mere  varieties.  They  are  without  exception  inhab- 
itants of  salt-water.  In  large  rivers,  such  as  the  Elbe, 
phosphorescing  vibrios  have  been  found  which,  otherwise, 
resemble  the  vibrio  of  Asiatic  cholera.  It  is  possible  that 
these  vibrios  are  derived  from  the  sea.  All  the  phosphor- 
escing bacteria  known  are  aerobic  and,  with  possibly  one 
exception,  are  non-pathogenic.  They  are,  with  the  excep- 
tion of  one  micrococcus  and  the  vibrios  mentioned,  all  rod- 


118  BACTERIOLOGY. 

shaped  bacteria.  In  order  to  give  expression  to  their 
characteristic  physiological  property,  they  have  been 
grouped  together  under  the  generic  term  photobacterium. 

The  property  of  phosphorescing  is  even  more  unstable 
than  that  of  pigment-production.  Nearly  all  of  the  organ- 
isms readily  lose  their  phosphorescence  when  grown  for 
some  time  upon  artificial  media.  This  is  chiefly  due  to  the 
reaction  and  composition  of  the  ordinary  media.  As  indi- 
cated above,  the  natural  habitat  of  these  organisms  is  the 
sea,  and  they  require,  consequently,  a  large  amount  of 
salt.  About  4  per  cent,  of  common  salt  should  be  added  to 
the  medium,  which  is  still  further  improved  by  the  addition 
of  small  amounts  of  glycerin  and  asparagin.  The  formula 
for  such  a  medium  will  be  found  under  B.  phosphorescens. 
On  this  medium,  the  phosphorescence  develops  to  a  remark- 
able degree.  Some  species  which  have  been  grown  in  the 
laboratory  for  some  years  and  apparently  have  lost  all 
power  of  phosphorescing,  promptly  develop  this  property 
when  grown  on  such  media. 

It  is  quite  possible  that  bacteria  will  be  found  inland 
which  ordinarily  do  not  phosphoresce  but  may  do  so  when 
developed  on  media  which  are  rich  in  salt.  The  author  has 
observed  in  Ann  Arbor  a  splendid  phosphorescence  ou 
kidneys.  The  composition  of  these  organs  might  favor 
the  development  of  such  otherwise  non-phosphorescir-g 
bacteria.  Again,  the  glow  observed  at  times  on  manure 
heaps  may  possibly  be  due  to  similar  forms  favored  by  the 
large  amount  of  saline  constituents. 

Absence  of  oxygen  and  an  acid  reaction  are  unfavora- 
ble to  the  production  of  light.  The  temperature  of  the 
body  is  likewise  unfavorable.  The  very  best  phosphor- 
escence is,  as  a  rule,  obtained  when  the  growth  takes  place 
at  about  15°.  Gelatin  tubes  and  plates  under  these  condi- 
tions will  phosphoresce  for  more  than  a  month.  The  phe- 
nomenon of  phosphorescence  is  observed  at  0°,  or  even  at  a 
lower  temperature.  On  a  cold  night  the  colonies  on  a  plate 


THE    CHEMISTRY  OP    BACTERIA.  119 

shine  like  the  stars  in  the  heavens  and  when  very  numerous 
a  diminutive  "  milky  way"  will  be  seen. 

The  character  of  the  light  emitted  by  these  bacteria 
varies  somewhat  for  the  various  species,  but  not  enough  to 
be  of  use  in  distinguishing  between  them.  Moreover,  the_ 
light  will  vary  according  to  the  composition  of  the  soil. 
It  may  be  whitish,  bluish  or  greenish.  The  intensity  of  the 
light,  at  times,  is  such  that  a  streak  culture,  on  inclined 
fish-gelatin,  is  sufficient  to  reveal  the  time,  on  a  watch.  In- 
deed, photographs  of  a  watch,  fish,  etc.,  have  been  made 
by  means  of  such  light.  When  examining  cultures  for  phos- 
phorescence it  is  essential  that  the  eye  should  be  accus- 
tomed to  darkness,  in  order  to  perceive  the  delicate  bacterial 
light.  At  night  time  the  phosphorescence  can  be  recog- 
nized, at  once,  by  any  one.  In  the  day  time,  the  transition 
from  bright  day-light  to  total  darkness  is  so  sudden  that  it 
will  be  necessary  to  wait  for  some  minutes  before  the  phos- 
phorescence will  be  perceived. 

Very  little  that  is  positive  can  be  said  regarding  the 
mechanism  of  phosphorescence.  The  commonly  accepted 
view  is  that  the  emission  of  light  is  the  result  of  intracel- 
lular  activity.  As  long  as  the  protoplasm  of  the  cell  is  at 
work  it  liberates  energy.  This  is  ordinarily  set  free  as 
heat,  but  in  these  organisms  it  is  supposed  that  the  energy 
or  wave  motion  is,  in  part  at  least,  that  which  corresponds 
to  light.  On  the  other  hand,  the  striking  similarity  be- 
tween pigment  production  and  phosphorescence  should  not 
be  overlooked.  Thus,  free  access  of  oxygen  is  essential  to 
both  phenomena;  high  temperatures  are  unfavorable  and 
even  give  rise  to  atypical  species  or  varieties;  low  temper- 
atures are  especially  favorable  to  the  production  of  pigment 
and  of  light.  In  the  latter  case,  both  may  remain  bright 
and  unaltered  after  the  lapse  of  many  weeks.  It  would 
seem,  therefore,  that  phosphorescence,  like  color,  is  asso- 
ciated with  the  production  of  certain  bacterial  products 


120  BACTERIOLOGY. 

which  are  made  within,  but  eliminated  from  the  cell.  The 
fact  that  filtered  cultures  of  phosphorescing-  bacteria  fail 
to  give  off  light  does  not  disprove  this  view.  The  phos- 
phorescing- substance,  like  most  of  the  bacterial  pigments, 
may  be  insoluble  in  water.  It  undoubtedly  is  very  unstable 
since  any  chemical  treatment  yields  inert  products.  Vari 
ous  aldehyde  derivatives  are  known  to  phosphoresce  in 
alkaline  solutions,  and  it  is  possible  that  similar  products 
are  elaborated  by  certain  bacteria. 

Heat   Production. 

The  heat  of  the  animal  body  is  ascribed  to  the  oxida- 
tion or  combustion  of  the  assimilated  food.  In  the  cleav- 
age of  complex  bodies  heat  is  almost  invariably  generated. 
Inasmuch  as  in  the  various  fermentations,  the  process  is  es- 
sentially one  of  cleavage,  it  follows  that  an  evolution  of 
heat  must  be  expected.  It  matters  little,  whether  the  fer- 
mentative change  is  induced  by  bacteria,  yeasts,  or  by 
moulds,  in  either  case  heat  will  be  produced.  Several  in- 
stances may  be  mentioned  to  show  to  what  extent  thermo- 
genic  bacteria  may  raise  the  temperature  of  the  medium  in 
which  they  grow. 

In  ordinary  alcoholic  fermentation,  due  to  the  yeast- 
plant,  the  temperature  of  the  liquid  may  rise  to  an  appre- 
ciable extent.  It  is  well  known  that  turnips,  potatoes,  etc., 
especially  if  moist,  when  placed  in  large  heaps  in  a  closed 
room  will  warm  up  to  50  or  60°,  as  a  result  of  fermentation. 
A  strong  odor  of  tri-methylamin  will  be  recognized  and 
many  of  the  tubers  will  show  an  evolution  of  gaseous  pro- 
ducts. Similar  spontaneous  heating  is  observed  at  times 
in  masses  of  hops  and  the  same  fish-odor  is  present. 

Another  interesting  case  is  met  with  in  the  curing  of 
tobacco.  After  a  preliminary  drying  the  leaves  are  packed 
in  large  masses  and  subjected  to  a  fermentation.  The  tern- 


THE    CHEMISTRY   OF    BACTERIA.  121 

erature  in  the  interior  of  the  mass  rises  and,  in  a  day  or 
two,  may  reach  50°  or  more.  As  a  result  of  this  fermenta- 
tion a  special  flavor  or  aroma  is  imparted  to  the  tobacco. 
Inasmuch  as  this  fermentation  is  due  to  certain  bacteria,  it 
has  been  proposed  to  utilize  pure  cultures  of  certain  of 
these  organisms  in  order  to  obtain  good  grades  out  of 
otherwise  poor  material.  In  the  preparation  of  snuff, 
the  tobacco  is,  likewise,  subjected  to  a  fermentation,  in 
which  the  temperature  rises  even,  higher  than  mentioned 
above. 

In  the  preparation  of  sauer-kraut,  fermented  hay,  and 
ensilage,  similar  phenomena  take  place.  The  material  is 
thoroughly  stamped,  or  compressed,  in  large  masses,  and 
allowed  to  ferment.  Not  infrequently,  the  temperature  on 
the  inside,  will  rise  to  70°.  The  material  becomes  acid,  due 
largely  to  the  presence  of  lactic,  and  butyric  acids.  At  the 
same  time  it  becomes  soft,  and  acquires  a  more  or  less, 
characteristic,  not  unpleasant  odor. 

The  high  temperature  which  develops  in  the  interior 
of  large  masses  of  green  hay,  may,  if  air  is  suddenly  admit- 
ted, lead  to  spontaneous  combustion.  This  may  also  occur 
with  masses  of  moist  cotton,  and  the  fires  in  cotton  laden 
ships  are  not,  infrequently,  due  to  such  causes. 

Toxicogenic  and  Pathogenic  Bacteria. 

In  fermentation  and  putrefaction,  more  or  less  com- 
plex, dead  animal  and  vegetable  substances  are  acted  upon 
by  diverse  organisms,  such  as  bacteria,  moulds  and  yeasts. 
They  are  transformed  into  relatively  simpler  compounds, 
and  eventually  into  inorganic  forms,  such  as  carbonic  acid, 
ammonia,  nitrites,  nitrates,  sulphates,  phosphates,  etc., 
which,  as  shown  heretofore,  are  then  utilizable  by  living 
plants.  Hence,  lifeless  remains  are  indispensable  to  new 
life,  and,  the  micro-organisms  which  accomplish  this 


122  BACTERIOLOGY. 

change,  are  absolutely  essential  to  the  continued  existence 
of  higher  plants  and  animals. 

The  products,  elaborated  by  the  majority  of  bacteria, 
are  practically  harmless,  if  introduced  into  the  living-  body. 
It  follows,  therefore,  that  the  vast  majority  of  bacteria  are 
unable  to  produce  poisoning-,  or  disease,  in  man  or  in  ani- 
mals. A  relatively  small  number  of  bacteria  give  rise  to 
poisonous  products,  and  are  also  able  to  grow  in  the  living 
body.  In  such  cases  they  live  at  the  expense,  and  to  the 
injury  of  the  host,  and  hence  induce  disease.  Such  organ- 
isms are,  therefore,  designated  as  pathogenic.  These  will 
receive  special  consideration  in  subsequent  chapters. 
There  are  bacteria  which  produce  poisonous  products,  but 
cannot  grow  in  the  living  body.  When  such  organisms  de- 
velop in  foods  they  may  give  rise  to  poisonous  products, 
which  will  cause  intoxication  when  the  food  is  consumed. 
The  subject  of  poisonous  foods  is  briefly  touched  upon  in 
Chapter  XV1. 

The  term  toxicogenic  applies  to  all  bacteria,  which  pro- 
duce poison,  irrespective  as  to  whether  they  are  able,  or 
unable,  to  grow  in  the  living  body. 

1  Additional  information  will  be  found  in  Vaughan  and  Novy, 
Ptomai'ns  and  Leucomains,  3rd  ed. 


CHAPTER    VI. 


THE    MICROSCOPE.— THE    HANGING   DROP.— SIMPLE    STAINING. 


In  view  of  the  fact  that  many  of  the  students  begin  the 
study  of  bacteriology  without  any  previous  experience  in 
the  use  of  a  microscope,  it  is  very  desirable  to  describe  this 
instrument,  and  the  manner  in  which  it  should  be  employed. 

It  is  customary  to  speak  of  simple,  and  of  compound  mi- 
croscopes. The  former  consist,  usually,  though  not  neces- 
sarily, of  a  single  lens,  as  in  the  case  of  an  ordinary  read- 
ing glass.  In  the  simple  microscope,  the  rays  of  light 
which  enter  the  eye 
come  directly  from  the 
object,  and  a  virtual 
image  is  produced. 
Fig.  15  illustrates  the 
action  of  a  simple  mi- 
croscope. It  should 
be  observed  that  the 
object  is  between  the 
principal  focus  P  and 

FIG.  15.    Virtual  image,  simple  microscope  (Carpenter). 

the  lens.      This  figure 

also  illustrates  the  action  of  the  eye-piece  in  the  compound 

microscope. 

If  the  object  is  placed  beyond  the  principal  focus  (P), 
as  in  Fig.  16,  a  real  image  will  result,  and  can  be  received 
on  a  screen.  This  corresponds  to  the  action  of  the  objec- 
tive in  the  compound  microscope. 

The  compound  microscope  consists  of  two  set  of  lenses, 
the  objective  and  the  eye-piece.  The  former  is  placed  near 
the  object,  and  gives  rise  to  a  real  image  (Fig  17  A  B).  This 


124 


BACTERIOLOGY. 


image,  inverted  and  reversed,  lies  inside  of  the  principal 
focus  of  the  eye-piece,  and  the  rays  of  light  leave  it  as  if 
they  came  from  a  real  object.  If  a  screen  was  placed  at 
this  point,  it  would  show  an  image.  Since  this  image  lies 
inside  of  the  principal  focus,  it  becomes  magnified  by  the 
eye-piece  (E),  and  the  virtual  image  (C  D)  is  produced.  It 
has  been,  not  inaptly,  said  that  "  a  compound  microscope  is 
a  simple  microscope  applied,  not  to  the  object,  but  to  its 
image  already  magnified  by  the  first  lens." 


FIG.  16.    Real  image  (Carpenter). 

The  single  lens  naturally  represent  the  earliest  form  of 
the  microscope.  The  English  monk,  Roger  Bacon,  in  1276, 
appears  to  have  been  the  first  to  recognize  the  peculiar 
properties  of  a  lens.  It  was  he  who  applied  the  new  knowl- 
edge to  the  construction  of  spectacles.  It  was  not,  how- 
ever, till  the  beginning  of  the  17th  century,  that  the  micro- 
scope was  sufficiently  perfected  to  bring  about  the  discov- 
ery of  the  existence  of  microscopic  life.  The  discovery  of 
the  compound  microscope  may  be  said  to  have  been  made 
by  Galileo  in  1610,  although  it  is  probable  that  others  may 
have  antedated  him  by  a  few  years.  The  early  compound 
microscope  consisted  of  a  single  lens  for  an  objective,  and 
of  another  single  lens  for  an  eye-piece.  Such  an  instru- 
ment, necessarily  gave  very  imperfect  results. 

The  image  produced,  by  a  single  lens,  is  not  a  perfect 
reproduction  of  the  original  object.  All  the  rays  of  light 
do  not  meet  in  the  same  plane,  and  hence,  the  image  has  a 
spherical  appearance.  This  fault  of  a  lens  is  known  as 
spherical  aberration.  Again,  the  lens,  acting  as  a  prism,  de- 
composes some  of  the  rays  of  light  which  pass  through  it. 


THE    MICROSCOPE. 


125 


The  violet  component  is  bent  most,  and  hence,  is  brought 
to  a  focus  at  a  different  point  from  the  red  ray,  which  is 
bent  the  least.     The  result  is 
a  fringe  of   colors.      This   is 
designated  as  chromatic  aber- 
ration. 

It  is  necessary,  therefore, 
to  correct  the  chromatic  and 
spherical  aberration,  in  order 
to  obtain  the  best  optical  re- 
sults. The  spherical  aberra- 
tion is  partially  corrected  by 
means  of  stops  or  diaphragms, 
which  hold  back  the  peri- 
pheral rays,  and  allow  only 
the  central  ones  to  pass 
through,  since  these  give  rise 
to  an  almost  flat  image.  Such 
diaphragms  are  shown  in  Fig. 
17.  The  use  of  flint  glass  still 
further,  serves  to  correct  this 
error. 

The  chromatic  aberration 
is  more  difficult  to  correct. 
The  first  successful  attempt 
at  the  correction  of  chro- 
matic aberration  in  an  objec- 
tive, was  made  by  an  Italian, 
Marzoli,  in  1811.  In  other 
words,  200  years  elapsed  be- 
tween the  discovery  of  the 


FIG.  17.  Principle  of  the  compound  mi- 
croscope (Carpenter).  F— Object  in  focus, 
above  this  an  objective  with  diaphragm; 


..  _,      ..          above  tins  an    objective  with  diapnrag 

Compound  miCrOSCOpe  and  the     AB-Real  image  of  F,  in  opening  of  d 


phragm;  above  this  a  compensation  ocular 
which  magnifies  the  real  image  AB,  thus 
forming  the  virtual  image  CD. 


correction  of  the  most  serious 

defect  in  the  working  of  the 

objective.     This  early  work,  however,  seems  not  to  have 

attracted  much  attention,  and  Chevalier,  of  Paris,  was  given 


126  BACTERIOLOGY. 

credit  for  introducing",  in  1825,  the  method  of  correcting 
chromatic  aberration.  The  real  value  of  a  microscope,  as 
an  aid  in  scientific  research,  dates  from  this  period.  These 
improved  objectives,  since  the  color  defects  were  done  away 
with,  were  designated  as  achromatic  objectives.  This 
achromatism  was  obtained  by  the  combination  of  two 
glasses,  crown  and  flint. 

The  dispersive  power  of  flint  glass  is  almost  double  that 
of  crown  glass,  and  hence,  a  crown  bi-convex  lens  is  fitted 
into  a  plano-concave  lens  of  flint  glass.  The  effect  of  this 
combination  will  be  understood,  if  a  prism  of  crown  glass, 
having1  an  angle  of  50°,  be  placed  by  the  side  of  an  inverted 
one  of  flint  glass,  having-  an  angle  of  25°.  The  ray  of  lig-ht 
passing-  throug-h  the  crown  glass  is  decomposed  into  its 
components,  the  colored  rays  of  the  spectrum.  These  then 
enter  the  inverted  flint  glass  prism,  and  are  reconstituted 
to  white  light.  The  concave  flint  lens  is  virtually  an  in- 
verted prism,  with  reference  to  the  convex,  crown  glass  lens. 
Refraction  is  thus  obtained  without  dispersion. 

Even  the  best  achromatic  objective  gives  an  image 
that  is  not  wholly  free  from  color.  This  is  due  to  the  fact 
that  the  two  kinds  of  glass  employed  do  not  disperse  the 
several  rays  in  the  same  proportion.  ^For  instance,  the 
green  ray  may  occupy  the  exact  middle  of  the  spectrum 
produced  by  one  glass,  whereas,  in  that  produced  by  the 
other,  it  may  lie  to  one  side,  possibly  only  one-third  of  the 
distance  from  the  red  to  the  violet.  If,  therefore,  the  red 
and  violet  rays  are  neutralized,  the  green  will  still  remain. 
Consequently,  there  is  always  a  certain  amount  of  color 
present  around  the  image  of  an  achromatic  objective.  This 
is  known  as  the  secondary  spectrum. 

About  10  years  ago  Abbe  and  Zeiss  prepared  special 
kinds  of  glass,  the  so-called  borate  and  phosphate  glass, 
by  means  of  which  it  was  possible  to  do  away  with  the 
secondary  spectrum.  These  objectives,  which  represent 


THE  MICROSCOPE.  12T 

the  highest  optical  attainment  at  the  present  time,  are  de- 
signated as  apochromatic.  The  lenses,  entering-  into  the 
composition  of  these  objectives,  are  made  of  the  special 
glasses  mentioned,  and  in  addition,  there  is  present  a  lens 
made  of  fluorite. 

The  employment  of  a  cover-glass,  between  the  object 
and  the  objective,  disturbs  the  correction  of  the  latter. 
The  rays  of  light,  passing  from  the  object  through  the 
cover-glass,  are  refracted,  so  that  the  peripheral  rays 
which  enter  the  objective,  seem  to  come  from  a  point 
nearer  the  objective  than  do  the  central  rays.  This  condi- 
tion is  overcome  by  making  an  under-corrected  objective, 
which  will  focus  both  these  points  at  once.  In  objectives 
provided  with  a  "collar"'  under-correction  is  obtained  by 
turning  the  collar  so  as  to  bring  the  back  lenses  nearer  to- 
the  front  lens.  The  correction  collar  is  placed  only  on  the 
dry,  high  power  objectives.  In  the  absence  of  a  collar,  the 
necessary  correction  for  a  thick  cover-glass  can  be  obtained 
by  shortening  the  tube-length,  thus  causing  the  eye-piece 
to  approach  the  objective.  Objectives  are  usually  made 
without  a  collar,  and  are  corrected  for  a  cover-glass  having 
a  thickness  of  0.17  mm.  Markings  on  an  object,  which  are 
easily  seen  when  a  cover-glass  of.  this  thickness  is  em- 
ployed, disappear  when  an  appreciably  thicker,  or  thinner 
cover-glass  is  used.  Unlike  the  dry  lenses,  the  oil  immer- 
sion objectives  are  largely  independent  of  the  thickness  of 
the  cover-glass. 

It  is  evident  that  the  objective  is  the  most  important 
pt'irt  of  the  microscope.  Consequently,  in  purchasing  a 
microscope,  special  attention  should  be  given  to  the  quality 
of  the  objectives.  A  good  microscope  means,  above  all, 
good  objectives.  It  is  a  common  error  for  the  student  to 
believe  that  the  best  microscope  is  the  one  that  magnifies 
the  most.  This  is  far  from  being  true.  It  is  not  the  power 
of  magnification,  but  the  ability  to  show  up  objects  in  their 


128  BACTERIOLOGY. 

minutest  details  which  makes  an  instrument  truly  valuable. 
The  properties  which  a  good  objective  should  possess  are 
as  follows: 

1. — A  proper  magnifying  poiver. — In  the  case  of  a  single 
lens  the  magnifying  power  may  be  said  to  depend  upon  its 
focal  distance.  The  shorter  the  focal  length  the  higher  the 
magnification.  Therefore,  a  lens  with  a  one-fourth  inch 
focus  will  magnify  more  than  one  having  a  focal  distance 
•of  one  inch.  In  the  case  of  the  objective,  we  have  to  deal 
not  with  one  lens,  but  with  a  combination  of  lenses,  and  it 
is  customary  then  to  speak  of  the  "equivalent  focal  dist- 
ance." This  represents  what  would  be  the  focal  distance 
of  a  simple  lens  having  the  same  magnification  as  the  ob- 
jective. When,  therefore,  a  lens  is  designated  as  a  one- 
fourth  inch  objective,  it  does  not  mean  that  the  dist- 
ance from  the  object  to  the  front  lens  is  one-fourth  of  an 
inch.  On  the  contrary,  this  "working  distance,"  as  it  is 
called,  would  be  much  less  than  a  fourth  of  an  inch.  The 
one-fourth  inch  objective,  however,  does  possess  the  same 
magnification  as  a  single  lens  having  a  focal  distance  of 
one-fourth  of  an  inch. 

A  table  showing  the  magnifying  power  of  the  several 
objectives  and  eye-pieces  is  usually  supplied  by  the  instru- 
ment maker.  The  magnifying  power,  of  a  given  objective 
and  eye-piece,  can  be  obtained  by  the  following  simple 
method.  A  stage  micrometer,  or  object  of  known  length,  is 
necessary.  The  micrometer  is  a  glass  slide  on  which  an 
accurate  scale  has  been  cut.  The  scale  may  consist  of 
one  mm.  divided  into  100  parts.  The  micrometer  is  ad- 
justed on  the  stage  of  the  microscope.  The  draw-tube  of 
the  latter  should  be  drawn  out,  vertically,  so  that  the 
upper  surface  of  the  eye-piece  is  10  inches  from  the  table. 
On  looking  into  the  microscope,  both  eyes  kept  open,  the 
image  of  the  micrometer  will  be  seen  to  be  projected  on  the 
table.  The  limits  of  the  projected  scale  can  be  marked  on 
the  table,  and  the  distance  then  measured.  If,  for  exam- 


THE  MICROSCOPE.  129 

pie,  one  mm.  is  magnified  so  as  to  measure  100  mm. ,  it  is 
evident  that  this  magnification  of  the  objective  and  eye- 
piece is  100. 

The  student  should  distinguish  between  linear  and 
superficial  magnification.  The  former  is  always  meant  in 
scientific  work,  whereas,  the  latter  is  employed  for  popu- 
lar purposes.  As  an  example,  corresponding  to  the  above, 
a  one  mm.  square  would  be  magnified  to  a  100  mm.  square. 
The  linear  magnification  would  be  100;  the  superficial,  or 
area  magnification  would  be  10,000. 

2. — A  good  defining  power. — It  is  obvious  that  a  good 
objective  should  give  as  flat  a  field  as  possible.  Perfect 
flatness  of  the  field  is  not  attainable,  but  a  great  deal  can 
be  done  by  reducing  the  spherical  aberration.  The  defini- 
tion of  an  objective  is  likewise  increased  by  properly  cor- 
recting the  chromatic  aberration.  Moreover,  the  perfect 
centering  of  the  lenses  is  necessary  to  the  good  perform- 
ance of  an  objective.  The  flatness  of  field  can  best  be 
tested  by  a  stage  micrometer,  whereas  the  definition  proper 
can  be  tested  by  means  of  stained  bacteria,  such  as  the 
tubercle  bacillus. 

3. — A  good  resolving  and  penetrating  power. — By  the  re- 
solving or  delineating  power  is  meant  the  ability  of  a  lens 
to  show  up  fine  markings  and  delicate  structures.  A  normal, 
unaided  eye  may  recognize  divisions  on  a  scale  which 
are  rb  of  an  inch  apart.  Another  eye  may  fail  to  resolve 
these  lines,  whereas  a  third  may  be  able  to  recognize  even 
a  smaller  fraction  of  an  inch.  Again,  on  account  of 
"acuteness  of  vision",  one  person  may  make  out,  with 
the  unaided  eye,  a  double  star,  which  another  person 
may  fail  to  do.  The  resolving  power,  therefore,  and  not 
its  magnifying  power,  is  what  imparts  value  to  an  objec- 
tive. 

The  resolving  power  of  an  objective  depends  primarily 
upon  the  amount  of  light  which  it  admits.  The  light  that 
enters  is  included  in  the  angle  made  by  the  two  extreme 


130  BACTERIOLOGY. 

rays,  which  leave  the  object  (when  in  focus)  and  enter  the 
front  lens.  This  angle,  known  as  the  angle  of  aperture,  is 
shown  at  a,  in  Fig.  18.  This  figure  shows,  in  cross-section, 
the  lens  system  employed  in  the  construction  of  an  oil 
immersion  objective.  The  rays  of  light  diverging  from 
the  object  enter  the  front  of  the  small  lens.  It  is  obvious 
that  the  more  light  admitted  by  an  objective  the  more  dis- 
tinct the  image.  The  angle  of  aperture, 
therefore,  conditions  the  resolving  power  of 
an  objective. 

In  actual  practice,  the  rays  of  light, 
after  they  leave  the  object,  pass  into  the 
cover-glass  (a  denser  medium)  and  from  this 
into  the  air,  which  is  less  dense.  The  result 
is  refraction,  and,  more  or  less,  loss  of 
\«/  light  by  reflection.  If  the  air  is  replaced, 

FIG.  18.    Arrange-  by  a  denser   medium,  there  will  be  less  re- 

ment  of  lenses  in  an 

one-twelfth  inch  oil  fraction  and  hence,  it  is  possible  to  utilize 

immersion    objective 

(Carpenter),  a— An-  SOme  of  the  light  that  would  otherwise  be 

gle  of  aperture. 

lost.  These  considerations  led  to  the  intro- 
duction by  Amici,  of  the  water  immersion  objective.  In 
this  objective  a  drop  of  water  was  placed  between  the 
cover-glass  and  the  front  lens.  The  ray  of  light  passing 
from  the  cover-glass  into  the  water  suffered  less  refrac- 
tion than  it  would  if  it  passed  into  air.  Since  the  index 
of  refraction  of  water  is  1.0  and  that  of  crown  glass  is 
1.52,  it  is  evident  that  there  was  still  room  for  improve- 
ment. In  1878,  Stephenson  suggested  that  the  water  in 
the  immersion  objectives  be  replaced  by  an  oil  having 
the  same  index  of  refraction  as  crown  glass.  Abbe  and 
Zeiss,  thereupon,  introduced  the  homogeneous  oil  immersion 
objective.  The  cedar  oil,  which  is  placed  between  the 
cover-glass  and  the  front  lens  of  the  object,  has  the 
same  index  of  refraction  as  crown  glass  (1.52).  Conse- 
quently, a  ray  of  light  after  it  once  enters  the  cover-glass 
passes  in  a  straight  line  directly  into  the  objective.  The 


THE  MICROSCOPE.  131 

angle  of  aperture  is  utilized  to  its  widest  extent,  and,  as  a 
result,  the  resolving1  power  is  increased. 

The  resolving  power  of  an  objective,  therefore,  depends 
upon  the  angle  of  aperture  and  on  the  index  of  refraction 
of  the  medium,  which  is  between  the  cover-glass  and  the 
front  lens.  Abbe'  has  reduced  these  factors  to  a  mathe- 
matical expression,  which  is  known  as  the  numerical  aper- 
ture. The  numerical  aperture  of  an  objective  corresponds 
to  the  sine  of  half  the  angle  of  aperture,  multiplied  by  the 
index  of  refraction  of  the  medium  that  lies  between  the 
front  lens  and  the  objective.  It  is  usually  expressed  as, 

N.A.  =  n  sine,  u. 

n  represents   the   index   of     refraction,  and   u  represents 
half  the  angle  of  aperture. 

It  is  well  to  repeat,  that,  the  value  of  an  objective  de- 
pends not  upon  its  magnification,  but  upon  its  resolving 
power,  and  that  this  is  directly  proportional  to  its  numerical 
aperture.  Of  the  lenses  having  the  same  magnifying 
power,  the  one  that  has  the  highest  numerical  aperture  is 
the  most  valuable  and  the  most  expensive. 

For  ordinary  work,  it  is  not  desirable  to  have  an  oil  im- 
mersion objective  of  more  than  about  1.30  N.A.  since  above 
this,  on  account  of  their  peculiar  construction,  they  are  very 
liable  to  suffer  injury.  Zeiss  has  succeeded  in  making  ob- 
jectives possessing  a  N.A.  of  1.50,  where  the  theoretical 
limit  is  1.52.  By  means  of  fluorite  lenses,  and  a  special  im- 
mersion fluid,  mono-brom  naphthalen,  Zeiss  has  been  able 
to  produce  a  lens  having  a  N.A.  of  1.63,  which  represents 
the  highest  achievement  in  the  construction  of  objectives. 

The  penetrating  power,  or  depth  of  focus,  is  expressed  by 
^-^  In  other  words,  it  is  reduced  by  increasing  the  aperture, 
as  well  as  the  magnification.  Just  as  the  resolving  power 
is  necessary,  in  order  to  bring  out  the  structure,  or  lines  in 
a  given  plane,  so  a  penetrating  power  is  desirable,  in  order 
to  perceive  depth  in  an  object.  For  ordinary  work,  there- 
fore, it  is  preferable  to  employ  a  low  objective,  with  a  mod- 


132  BACTERIOLOGY. 

erate  aperture.     The  high  objective,  with  a  wide  aperture, 
is  to  be  used  only  as  the  special  occasion  requires. 

In  England  and  America,  it  is  the  custom  to  designate  objectives 
by  their  equivalent  focal  distance.  On  the  Continent,  however,  no 
such  designation  exists  and  some  makers  mark  the  objectives  with 
numerals,  others  with  letters.  Leitz,  for  example,  marks  his  objec- 
tives with  numbers  from  1,  which  corresponds  to  a  If  inch,  to  10  which 
is  a  TV  water  immersion  objective.  Zeiss,  on  the  other  hand,  desig- 
nates the  achromatic  objectives  with  letters,  while  the  apochromatics 
are  designated  by  the  equivalent  focal  distance  expressed  in  mm.  The 
oil  immersion  lenses  are  designated  after  the  English  method  TV,  fa 
iV  inch.  The  objectives  which  have  met  with  most  favor  in  bacter- 
iological studies  are  the  3,  7  and  fa  The  No.  3  corresponds  to  a  t 
inch,  and  to  Zeiss'  A.  The  No.  7  is  about  the  same  as  a  i  inch,  while 
the  Zeiss  D  is  a  £  inch. 

The  eye-piece,  or  ocular,  serves  to  magnify  the  image 
made  by  the  objective.  The  form  commonly  employed,  is 
that  known  as  the  Huyghenian.  It  consists  of  two  lenses, 
a  lower  or  field-glass,  and  an  upper  or  eye-glass.  Both 
lenses  are  plano-convex.  The  lower,  broad  lens  serves  to 
refract  the  rays  of  light,  thus  bringing  the  image  within 
the  focus  of  the  eye-glass. 

When  apochromatic  objectives  are  employed,  it  is  nec- 
essary to  use  special  oculars,  known  as  the  compensa- 
tion eye-pieces.  The  eye-piece  represented,  in  section,  in 
Fig.  17,  p.  125  is  of  this  type. 

The  designation  of  eye-pieces  is  subject  to  variation,  as 
in  the  case  of  objectives.  Some  designate  by  letters,  others 
by  numbers.  In  this  country,  they  are  designated  in  the 
same  way  as  the  objectives,  that  is,  according  to  their 
equivalent  focal  distance.  Thus,  a  2-inch  eye-piece  would 
magnify  the  same  as  a  single  lens  of  2-inch  focus.  The  eye 
pieces  of  low  power  are  spoken  of  as  "  shallow,"  whil 
those  of  high  power  are  called  "  deep."  The  compensating 
eye-pieces  of  Zeiss  are  designated  by  their  amplification. 
Thus,  a  2  eye-piece  doubles  the  initial  magnification  of  the 
objective;  an  18  eye-piece  magnifies  the  image  18  times. 


d 

: 


THE    MICROSCOPE. 


133 


It  is  well  to  have  three  eye-pieces,  having-  an  equivalent 
focal  distance  of  50,  35  and  25  mm.  respectively.  This  cor- 
responds to  numbers  0,  2  and  4  of  Leitz,  and  2,  1£  and  1 
inch  eye-pieces  of  this  country.  As  a  rule,  the  low  eye- 
piece should  be  used  for  work.  The  higher  eye-pieces  may 
be  used,  when  it  is  desired  to  increase  the  magnification.  It 
should  be  remembered,  however,  that  the  eye-piece  magni- 
fies the  image,  and  hence,  any  imperfections  that  may  exist 
in  the  image.  On  account  of  eye-piece  magnification,  the 
field  becomes  darker  and  less  distinct.  It  is  preferable, 
therefore,  to  obtain  an  increased  magnification  by  changing 
the  objectives,  rather  than  by  the  use  of  deep  oculars. 
When  apochromatic  objectives,  are  used,  it  is  possible  to 
employ  even  the  deepest  compensation  oculars. 

The  Abbe  condenser. — This  is  a  most  important  accessory 
to  a  microscope,  and,  indeed,  it  should  not  be  wanting  on 
an  instrument  intended  for  bac- 
teriological work.  The  usual 
Abbe  condenser  is  chromatic, 
although  for  special  purposes,  as 
in  micro-photography,  an  achro- 
matic condenser  is  employed. 
The  ordinary  condenser  is  made 
of  two  lenses,  as  shown  in  Fig. 
19,  and  has  a  numerical  aperture 
of  1.20.  By  the  introduction  of 
a  third  lens,  the  aperture  is  in- 
creased to  1.40. 

The  aim  of  the  condenser  is  to  illuminate  the  object  as 
much  as  possible.  Without  a  condenser,  given  a  plane 
mirror,  an  object  will  be  illuminated  by  a  pencil  of  light 
corresponding  to  its  own  diameter.  This  may  be  sufficient 
for  a  low  power,  but  when  high  powers  are  used,  it  is 
evident  that  more  light  must  be  thrown  on  the  object  in 
order  to  see  distinctly.  By  means  of  the  condenser,  the 


FIG.  19.  Cross-section  of  the  Abbe 
condenser,  showing  convergence  of  the 
rays  of  light. 


134  BACTERIOLOGY. 

broad  pencil  of  light,  corresponding-  to  the  diameter  of  the 
front  lens  of  the  condenser,  is  brought  to  a  focus  at  about 
2  mm.  above  the  upper  spherical  lens.  An  object,  there- 
fore, placed  in  this  focus  will  receive  all  the  light  that 
enters  the  condenser.  In  order  to  obtain  the  best  results, 
the  condenser  should  always  be  focussed  on  the  object. 
When  parallel  light  is  employed,  the  reflected  light  from  a 
white  cloud,  the  focus  will  lie  nearest  to  the  plane  face 
of  the  upper  lens.  When  artificial  light,  as  that  of  a 
lamp,  is  employed,  the  rays  that  are  brought  to  the  con- 
denser are  divergent,  and  hence,  the  focus  will  be  farther 
away  from  the  upper  face  of  the  condenser.  The  correct 
adjustment  of  the  Abbe  condenser  should,  therefore,  re- 
ceive the  same  attention  as  the  proper  focussing  of  an 
objective.  The  plane  mirror  should  always  be  used  with 
the  condenser,  since  the  concave  mirror,  if  employed,  would 
give  off  converging  rays.  These  would  be  brought  to  a 
focus  so  close  to  the  upper  face  of  the  condenser,  that, 
owing  to  the  thickness  of  the  glass-slide,  it  would  not  be 
possible  to  bring  ihe  object  into  this  focus. 

The  Abbe  condenser  is  ordinarily  employed  with  its 
optical  axis  in  line  with  that  of  the  microscope.  It  thus 
gives  central  illumination.  The  iris  diaphragm  on  the 
more  expensive  instruments  can  be  placed  in  an  excentric 
position  by  means  of  a  thumb-screw,  and  can  be  rotated  in 
this  position  about  the  optical  axis.  Hence,  oblique  light 
can  thus  be  admitted  from  any  desired  direction. 

The  condenser  is  always  accompanied  by  an  iris  dia- 
phragm. This  enables  one  to  regulate  the  amount  of  light 
which  enters  the  condenser.  If,  for  instance,  an  unstained 
section  of  a  liver  is  placed  under  the  microscope,  and  care- 
fully focussed,  it  will  be  seen  perfectly,  provided  the  dia- 
phragm is  partly  closed.  Now,  if  while  looking  at  the  section 
through  the  microscope,  the  diaphragm  be  thrown  wide  open 
the  object  will  at  once  become  invisible.  The  excessive 
amount  of  light  coming  from  the  unrestricted  condenser 


THE    MICROSCOPE.  135 

renders  the  object  invisible — it  destroys  the  so-called  struc- 
tural image.  An  uncolored  object  is  rendered  visible,  because 
of  inequalities  in  the  reflection,  or  refraction  of  the  light,  as 
it  strikes  the  object.  On  the  other  hand,  a  stained  object 
is  visible  because  of  the  color.  The  color  image,  unlike 
the  structural  image,  is  practically  unaffected  by  the  light 
admitted  through  an  open  condenser. 

The  following  rule,  therefore,  should  govern  the  use  of 
the  condenser:  When  examining  unstained  preparations,  as  a 
hanging-drop,  the  diaphragm  should  be  restricted.  When  exam- 
ining stained  preparations,  the  diaphragm  should  remain  open. 
In  the  former  case,  the  diaphragm  should  be  restricted  to 
such  an  extent,  that  on  looking  into  the  microscope  with 
the  objective  in,  or  near  the  focus,  it  gives  a  dim  twilight. 
The  diaphragm  is  contracted  so  that,  at  most,  a  pin-head 
opening  exists.  When  the  specimen  is  stained,  the  dia- 
phragm remains  open.  It  is  not  advisable  to  open  the  dia- 
phragm to  the  widest  possible  limit,  since  a  stained  object 
is  rendered  more  distinct  if  the  diaphragm  is  slightly  con- 
stricted. 

In  order  to  secure  the  best  illumination  of  an  object,  it 
is  necessary  (1)  to  set  the  plane  mirror  at  the  best  angle  for 
reflection  of  the  light,  as  it  comes  from  a  white  cloud;  (2)  to 
adjust  the  iris  diaphragm,  according,  as  the  object  is  stained, 
or  colorless;  (3)  to  focus  the  condenser  on  the  object.  Di- 
rect sunlight  should  never  strike  the  mirror,  or  the  stage  of 
the  microscope. 

The  stand. — The  various  parts  of  the  stand  will  be  un- 
derstood best  by  reference  to  the  frontispiece.  The  draiv- 
tube  slides  up  and  down  in  the  body  of  the  microscope.  It 
is  graduated,  and,  when  the  instrument  is  about  to  be 
used,  the  draw-tube  should  be  raised  to  the  line  marked 
17,  provided  no  nose-piece  is  present.  The  upper  face  of 
the  eye-piece  is  then  170  mm.  distant  from  the  shoulder  of 
the  objective.  This  position  should  always  be  maintained 


136  BACTERIOLOGY. 

when  at  work,  because  the  lenses  have  been  corrected  for 
this  distance.  A  deviation  from  this  distance  will  cause 
deterioration  in  the  image  perceived.  Usually,  the  micro- 
scope is  provided  with  a  nose-piece,  which  is  10  mm.  thick. 
When,  therefore,  the  nose-piece  is  attached  to  the  micro- 
scope, the  draw-tube  should  be  raised  to  the  line  marked 
16  (160mm.) 

The  objective  is  always  moved  to,  or  from,  the  object 
by  means  of  the  coarse  adjustment.  This  is  a  rack  and  pin- 
ion, and  is  used  to  bring  the  objective  near  the  focus.  It  is 
not  used  for  "focussing,"  except  when  the  low  objective 
(No.  3)  is  used.  When  the  high  power  objective  is  brought 
as  near  to  the  focus  as  possible,  by  means  of  the  coarse  ad- 
justment, it  is  then  focussed  exactly,  by  means  of  the  fine 
adjustment.  The  latter  is  a  fine  micrometer  screw,  by  means 
of  which  the  entire  body  of  the  microscope  is  raised  or  low- 
ered. 

For  bacteriological  purposes,  the  microscope  should  as 
a  rule  be  kept  in  a  vertical  position.  It  is  manifestly  in- 
convenient to  examine  plates,  hanging-drops,  etc. ,  with  an 
inclined  instrument.  Although  a  triple  nose-piece  is  not 
necessary,  yet  it  is  a  very  desirable,  time-saving  addition 
to  a  microscope. 

Care  of  the  microscope. — There  is  no  instrument  placed 
in  the  hands  of  the  student  which  requires  as  careful  atten- 
tion. An  untidy  microscope  at  once  characterizes  the  work 
of  the  individual.  The  following  general  directions  should 
be  closely  observed  in  taking  care  of  the  instrument: 

1.—  The  microscope  should  always  be  taken  up  and  car- 
ried about  by  means  of  the  pillar  below  the  level  of  the 
stage.  A  chamois  skin  should  be  employed,  in  order  not 
to  soil  or  remove  the  lacquer.  It  should  never  be  lifted  by 
taking  hold  of  the  fine  adjustment  tube,  or  by  the  barrel. 

2. — All  unnecessary  contact  of  the  fingers  with  the  lac- 
quered parts  should  be  avoided.  If  finger-marks  have  been 


THE    MICROSCOPE.  137 

left,  they  should  be  removed  by  gentle  breathing1  on  the 
spot,  and  then  slightly  rubbing  with  clean,  washed  linen, 
or  with  a  piece  of  chamois  skin.  Alcohol  should  never  be 
applied  to  the  lacquered  surface,  since  it  will  remove  the 
lacquer. 

3. — The  milled  parts  on  the  objective,  eye-piece,  draw-- 
tube,   etc.,    should    be   handled   and   not   the  neighboring 
smooth  parts. 

4. — The  stage  of  the  microscope  should  be  kept  clean 
and  dry.  The  glass  slide,  or  plate,  should  never  be  placed 
on  the  stage,  unless  the  under  surface  is  known  to  be  dry. 

5. — The  mirror  should  be  kept  clean  and  free  from  dust. 
The  same  is  true  of  the  Abbe  condenser. 

6. — The  front  lens  of  the  objective  must  be  kept  per- 
fectly clean.  The  No.  3  and  No.  7  objectives  are  dry  lenses, 
and  should  never  be  allowed  to  touch  oil,  water,  gelatin  and 
the  like.  If  oil  has  touched  the  lens,  it  should  be  carefully 
removed  by  means  of  a  cloth  moistened  with  a  drop  of 
alcohol.  Gelatin  and  agar  can  best  be  removed  by  touch- 
ing with  a  cloth  dipped  in  clean,  warm  water.  The  rV  inch 
objective  is  an  oil  immersion  lens,  and  is  never  used  dry.  A 
drop  of  cedar  oil  is  placed  between  the  front  lens  of  this 
objective  and  the  clean,  dry,  upper  surface  of  the  cover- 
glass.  At  the  close  of  the  day's  work,  the  cedar  oil  must 
be  removed  from  the  front  lens  by  gently  touching  several 
times  with  a  well  washed  piece  of  old  linen,  or  with  a  soft 
tissue-paper. 

7N — The  eye-pieces  should  be  kept  clean,  free  from 
moisture  and  dust. 

8. — At  the  close  of  the  day's  work,  the  microscope 
should  be  carefully  inspected,  cleaned  wherever  it  is  nec- 
essary, and  placed  in  its  locker. 

Measurement  of  an  object. — In  this  connection,  it  will  be 
well  to  describe  the  method  pursued  in  measuring  the  size 
of  an  object.  It  is  necessary  to  have  a  stage  micrometer 


138  BACTERIOLOGY. 

aud  an  ocular  micrometer.  The  former  is  a  glass  slide  on 
which  a  scale  of  1  mm.  is  subdivided  into  100  parts.  The 
ocular  micrometer  is  placed  inside  the  eye-piece  on  the 
diaphragm,  by  unscrewing"  the  eye-lens  which  is  then  re- 
placed. It  lies,  therefore,  in  the  same  plane  as  the  image 
made  by  the  objective.  If  this  is  not  the  case  the  dia- 
phragm should  be  raised  or  lowered  till  the  plane  of  the 
ocular  micrometer  coincides  with  that  of  the  image.  It  is 
necessary,  first,  to  ascertain  for  each  objective  the  value  of 
a  division  of  the  ocular  micrometer. 

The  stage  micrometer  is  focussed,  first,  with  the  No.  3 
objective.  It  will  probably  be  found  that  10  divisions  on  the 
scale  in  the  eye-piece  cover  15.5  divisions  of  the  stage  micro- 
meter. 1  division  of  the  ocular  micrometer  corresponds, 
therefore,  to  1.55  divisions  on  the  stage  micrometer.  Since, 
each  of  the  latter  represents  OT  mm.,  it  follows  that  1  division 
on  the  scale  in  the  eye-piece  corresponds  to  \^  =  0.0155.  The 
TiJWth  part  of  a  mm.,  as  explained  on  p.  24,  is  known  as  a 
micron,  and  is  designated  by  the  letter  p..  The  micron  is 
approximately  s^uth  of  an  inch.  Therefore,  each  division 
on  the  ocular  micrometer,  used  in  connection  with  this  same 
eye-piece  and  objective,  corresponds  to  15.5  /*.  If,  now,  an 
object  is  placed  under  this  No.  3  objective  in  place  of  the 
stage  micrometer,  and  it  corresponds  to  10  divisions  on  the 
scale  of  the  ocular  micrometer,  it  is  evident  that  the  object 
measures  155  //.. 

The  "  micrometer  value  "  of  the  No.  7  objective  is  ascer- 
tained in  the  same  manner.  Thus,  if  50  divisions  on  the 
ocular  scale  correspond  to  18  divisions  on  the  stage  micro- 
meter, 1  division  of  the  former  will  correspond  to  0.36 
divisions  of  the  latter  =  £±  —  .0036  mm.  =3.6  //..  Again, 
in  case  of  the  iV  inch  objective,  50  divisions  on  the  eye-piece 
micrometer  correspond  to  8.6  divisions  of  the  stage  micro- 
meter. Hence,  1  division  of  the  former  corresponds  to  y£  = 
0. 172  of  one  division  of  the  latter.  Since  each  division  of 
the  latter  represents  ita  of  a  mm.,  one  division  of  the  eye- 


THE    MICROSCOPE.  139 

piece  micrometer  represents  ^y  =  .00172  mm.  =  1.72  //. 
If,  therefore,  a  bacillus  is  examined  with  this  objective,  and 
occupies  three  divisions  on  the  eye-piece  scale,  it  is  1.72  x  3 
or  5.16  ,'j.  long. 

When  the  micrometer  value  of  an  objective  is  once  as- 
certained, in  the  manner  described,  it  becomes  a  very  easy 
matter  to  determine  the  exact  size  of  an  object  by  means  of 
the  ocular  micrometer.  It  should  be  remembered,  however, 
that  this  micrometer  must  always  be  placed  in  the  same 
eye-piece  with  which  the  original  measurements  were  made. 
Likewise,  only  the  particular  objectives  tested  should  be 
employed  in  such  measurements.  The  draw-tube,  it  is  un- 
derstood, should  be  drawn  out  when  measuring  an  object, 
to  the  same  distance  as  when  the  micrometer  value  was 
determined  (160  mm.) 

When  an  ocular  screw-micrometer  is  employed,  owing 
to  its  height,  the  draw-tube  must  be  lowered,  so  that  there 
is  a  distance  of  170  mm.  between  the  upper  surface  of  the 
ocular  and  the  shoulder  of  the  objective.  The  value  of  a 
division  on  the  screw  is  ascertained,  for  each  objective,  by 
means  of  a  stage  micrometer  in  the  manner  described 
above. 

The  approximate  size  of  an  object  can  be  determined 
without  the  use  of  a  micrometer,  by  dividing  the  length  of 
the  image,  as  projected  on  the  table  (p.  128),  by  the  mag- 
nification of  the  objective  and  eye-piece  used.  The  latter 
is  obtained  in  the  manner  described  on  p.  128,  or  from  the 
table  supplied  by  the  maker  of  the  instrument. 

Draiving  an  object. — The  reproduction  of  objects  seen 
under  the  microscope,  constitutes  a  most  important  training 
of  the  student.  The  object  should  be  drawn,  so  as  to  have 
the  size  of  the  image  reproduced.  This  can  only  be  done 
by  projecting  the  image  on  the  drawing  paper,  and  then 
sketching  in  the  outline.  The  paper  is  placed  on  the  table, 
on  the  right  side  of  the  microscope.  On  looking  into  the 


140  BACTERIOLOGY. 

microscope,  with  the  left  eye,  the  image  of  the  object  will 
appear  on  the  paper,  and  can  be  readily  outlined.  The 
relative  size  of  different  objects,  seen  under  the  microscope, 
can  thus  be  faithfully  reproduced.  With  a  little  care  and 
patience,  very  satisfactory  results  will  be  obtained  in  a 
short  time. 

Various  optical  contrivances  have  been  devised  to  facil- 
itate the  drawing1  of  microscopic  objects.  The  camera  lu* 
cida  of  Abbe  is  especially  useful  for  this  purpose. 

Cover-Glasses. 


The  slides  and  cover-glasses,  employed  for  microscopic 
work,  must  be  rigidly  clean.  The  usual  method  of  clean- 
ing cover-glasses,  is  to  immerse  them  in  alcohol,  and  then, 
to  wipe  with  a  clean  cloth  till  dry.  Frequently,  cover- 
glasses  thus  treated,  although  apparently  clean,  will  not 
permit  the  spreading  of  a  drop  of  water  on  their  surface. 
This  thin  layer  of  fatty  matter  often  cannot  be  removed, 
even  by  treatment  with  sulphuric,  or  with  acetic  acid.  The 
cover-glass  may  be  passed  rapidly,  five  or  six  times,  from 
above  downward,  through  a  Bunsen  flame,  in  which  case 
the  heat  destroys  the  organic  matter  on  the  cover-glass. 
When  thus  treated,  a  drop  of  water,  placed  upon  that  sur- 
face of  the  cover-glass,  which  was  in  direct  contact  with 
the  flame,  can  be  spread  evenly  over  the  entire  surface.  It 
will  not  gather  into  small  droplets,  as  is  usually  the  case. 
The  objection  to  this  method  is,  that  many  of  the  cover- 
glasses  crack  during  heating. 

The  following  method  of  preparing  cover-glasses  will 
give  perfect  satisfaction.  The  cover-glasses  are  immersed 
in  alcohol,  and  are  then  wiped  dry,  and  as  clean  as  possi- 
ble. They  are  then  placed  in  an  Esmarch  or  Petri  dish, 
and  heated  in  a  dry-heat  sterilizer  at  170-180°,  or  higher, 
for  one  hour.  The  organic  matter  is  thus  subjected  to  de- 


THE  HANGING-DROP.  141 

structive  distillation;  the  cover-glasses  are,  as  a  result,  per- 
fectly clean,  and  they  remain  thus,  as  long  as  they  are  kept 
in  the  covered  dish.  It  is  advisable  to  clean  up  a  box  of 
cover-glasses  at  a  time.  The  Fresenius  iron  drying  plate, 
with  its  six  cups,  is  especially  convenient  for  student's  use. 
The  temperature  of  this  plate  can  easily  be  raised  to  200^ 
Only  very  rarely,  will  this  procedure  for  obtaining  clean 
cover-glasses,  fail.  In  such  cases,  an  additional  passage 
of  the  cover-glass,  two  or  three  times,  through  the  flame, 
will  give  a  suitable  surface.  The  droplet  of  water,  or  the 
bacterial  suspension,  should  be  placed  on  that  side  of  the 
cover-glass  which  touched  the  flame. 

The  cover-glass  should  always  be  handled  with  a  pair 
of  forceps.  The  ordinary,  slender,  narrow-pointed  forceps, 
are  well  adapted  for  this  purpose.  In  staining,  however, 
they  have  one  serious  disadvantage.  The  weak  blades 
compress  on  the  slightest  pressure,  and  form  a  capillary 
along  which  the  staining  reagent  readily  drains  from  the 
cover-glass.  Under  these  conditions,  the  hands  of  the  stu- 
dent stain  more  readily  than  does  the  specimen. 


FIG.  20.    Cover-glass   forceps   of    the    author,    a— Simple 
form;  b— Forceps  provided  with  clasp. 

The  forceps  devised  by  the  author,  and  shown  in  Pig.  20, 
are  extremely  convenient  in  handling  cover-glasses.  The 
lower  blade  is  flat  and  has  a  broad  end  (2  mm.  wide),  with  a 
thin,  sharp  edge.  The  upper  blade  is  narrow,  bent,  and 
terminates  in  a  point,  which,  when  the  forceps  are  shut,  rests 
close  to  the  end  of  the  lower  blade.  The  advantages  pos- 
sessed by  these  forceps  are,  that  it  enables  one  to  pick  up 


142  BACTERIOLOGY. 

cover-glasses  from  a  flat  surface,  and  that,  in  staining1,  no 
capillary  drainage  exists.  Moreover,  owing-  to  the  point- 
contact,  the  specimen  can  always  be,  washed  perfectly 
clean,  without  leaving-  the  unsightly  spot  so  often  caused 
by  a  broad  pointed  pair  of  forceps. 

The  above  forceps  can  be  obtained,  supplied  with  a 
clasp  (Fig1.  20  &),  as  in  the  case  of  Ehrlich's  forceps.  In  this 
form,  it  will  be  found  to  be  superior  to  the  awkward,  heavy 
Cornet  forceps,  or  its  several  modifications. 

Examination  of  Living  Bacteria.1 

This  is  intended  to  show  the  bacteria  in  their  natural 
condition.  The  study  of  an  organism  is  manifestly  incom- 
plete, if  the  examination  of  the  living  form  is  omitted.  A 
great  deal  of  information  can  thus  be  acquired,  which  might 
otherwise  be  overlooked.  Care  should  be  bestowed  espec- 
ially upon  the  form  and  grouping  of  the  cells,  the  presence 
or  absence  of  motion,  the  appearance  of  the  protoplasm, 
whether  colored  or  granular,  the  presence  of  sporogenic 
granules,  and  of  spores.  Obviously,  the  process  of  cell  di- 
vision, spore  formation,  and  germination  can  only  be  ob- 
served on  living  organisms. 

The  simplest  method  for  examining  living  bacteria  is  as 
follows:  A  little  of  the  growth  is  picked  up  on  the  end  of 
a  sterile,  cooled  platinum  wire  (Fig.  28  e),  which  is  then 
touched,  several  times,  to  a  drop  of  water  on  a  glass  slide. 
The  drop  of  water  should,  as  a  result,  show  a  visible  cloud- 
iness. It  is  then  covered  with  a  glass  slip,  and  examined 
under  the  microscope.  When  this  procedure  is  followed,  it 
is  desirable  that  the  drop  of  water  taken  be  so  small  that  it 
will  not  float  the  cover-glass. 

The  examination  of  the  living  organism  on  an  ordinary 
glass  slide,  in  the  manner  indicated,  is  not  always  satisfac- 

lfThe  examination  of  hanging-drops  and  the  staining-  of  bacteria 
follows  the  work  which  is  given  in  the  next  chapter. 


THE   HANGING-DROP.  14& 

tory,  because  the  liquid  soon  begins  to  evaporate  and,  as  a 
result,  rapid  currents  are  established.  To  overcome  this- 
evaporation,  it  is  customary  to  examine  the  material  in  a 
"  hanging-drop."  For  this  purpose,  a  thick  glass  slide,  hav- 
ing a  concave  well  in  the  middle,  is  made  use  of.  The  or- 
dinary "concave,"  or  hollow  slides,  usually  on  the  market,_ 
are  so  shallow  that  the  drop  of  water  will  touch  the  bottom. 
This  should  not  occur. 

The  "  hanging-drop "  is  easily  made  in  the  following- 
manner:  Place  a  small  drop  of  water  on  a  ^  inch,  clean 
cover-giass  which  is  placed  on  the  table  or  on  a  short 
board.  The  drop  must  be  small  enough  so  that  it  will 
not  run,  if  the  cover-giass  is  placed  on  edge.  The  growth 
from  the  potato,  or  other  medium,  is  then  touched  off  into  the 
drop  of  water,  by  means  of  a  sterile  platinum  wire.  A  ring 
of  vaselin  is  placed  around  the  edge  of  the  well  on  the  upper 
side  of  the  concave  slide,  by  means  of  a  brush  or  match 
stick.  The  slide,  with  its  ring  of  vaselin,  is  then  inverted 
over  the  cover-glass  and  gently  pressed  down  The  cover- 
glass  now  adheres  to  the  slide,  which  is  then  inverted. 
Care  should  be  taken  to  see  that  the  vaselin  is  continuous 
around  the  edge  of  the  well.  If  such  is  the  case,  no 
evaporation  of  the  drop  of  water  can  take  place,  and  hence, 
the  hanging-drop  can  be  examined  at  leisure  and  without 
the  presence  of  annoying  currents  in  the  liquid.  Fig.  21 
indicates  the  hanging-drop  in  longitudinal  cross-section. 


[  ^^  I 

FIG.  21.      The  "  hanging-drop,"  showing 
concave  slide  and  suspended  drop. 

The  Ranvier  slide  is  very  useful  for  certain  purposes. 
The  central  portion  of  the  slide  is  ground  down  so  that  it  is 
about  iV  mm.  below  the  surface.  This  shallow  well  is  sur- 
rounded by  a  deeper  groove.  The  drop  of  water,  instead  of 
being  convex  as  above,  is,  in  this  case,  flattened  out  into  a  thin 
layer.  The  edge  of  the  well  is  ringed  with  vaselin  as  above. 


144  BACTERIOLOGY. 

Examination  of  the  hang  ing -drop. — Place  the  slide,  with 
the  cover-glass  uppermost,  on  the  stage  of  the  microscope 
and  find  the  edge  of  the  drop  with  a  low  power — No.  3,  or 
frd  inch  objective.  If  too  much  light  is  present,  constrict 
the  diaphragm.  The  edge  of  the  drop  should  be  seen  as  a 
sharp  line  passing  through  the  field  of  the  microscope. 
Holding  the  slide  between  the  thumb  and  forefinger  of  the 
left  hand,  slowly  move  it  so  that  the  edge  of  the  drop  con- 
stantly remains  in  the  field.  In  this  way  the  entire  edge,  or 
circumference  of  the  drop,  should  be  examined,  chiefly  for  the 
purpose  of  practice  in  moving  a  slide  under  the  microscope. 
Owing  to  the  minute  size  of  bacteria,  they  cannot  be  seen 
under  this  magnification.  At  most,  fine  granules  can  be 
detected.  To  observe  the  individual  cells,  therefore,  recourse 
must  be  had  to  a  higher  power — No.  7,  or  £  inch  objective, 
or  to  the  Atli  inch  homogeneous,  oil  immersion  objective. 

Examination  with  the  No.  7  objective. — Having  found  the 
edge  of  the  drop  with  the  No.  3  objective,  replace  this  with 
No.  7,  by  rotating  the  nose-piece.  Then  lower  the  tube  of 
the  microscope  by  the  coarse  adjustment,  till  the  objective 
almost  touches  the  cover-glass.  The  field  of  the  micro- 
scope is  now,  usually,  very  dark;  hence  open  the  diaphragm 
a  trifle  to  admit  enough  light,  so  as  to  see  distinctly.  With 
the  fine  adjustment  now  raise  the  microscope  tube,  till  the 
edge  of  the  drop  is  brought  out  distinctly.  By  focussing 
the  edge  carefully,  the  bacteria  will  be  readily  detected. 
Now  move  the  slide,  as  mentioned  above,  so  as  to  examine 
the  entire  edge  of  the  drop,  and  also  the  center.  Study  the 
characteristics  of  the  micro-organisms  present,  especially 
with  reference  to  form,  structure  and  motility. 

In  working  with  a  high  power,  while  focussing,  it  is 
desirable  to  constantly  hold  the  slide  between  the  thumb 
and  forefinger  of  the  left  hand,  imparting  to  it  a  slight  mo- 
tion. If  this  motion  is  arrested,  it  is  due  to  pressure  of  the 
objective,  which  has  been  lowered  too  far,  and  unless  the 
pressure  is  promptly  relieved,  damage  may  result.  The 


STAINING  OF    BACTERIA.  145 

student  should  acquire,  as  soon  as  possible,  the  habit  of 
keeping-  both  eyes  open,  when  using  the  microscope. 


Examination  with  the  Mh  inch  homogeneous  oil  immersion 
objective.  —  Having-  studied  the  bacteria  in  the  hang-ing-  -drop 
with  the  No.  7  objective,  replace  the  No.  3  objective,  and 
ag-ain  find  the  edg-e  of  the  drop.  Now  raise  the  tube  of  the 
microscope,  by  means  of  the  coarse  adjustment,  and  bring 
the  TVth  inch  objective  into  position.  Place  a  drop  of  cedar 
oil  on  the  center  of  the  cover-glass,  and  lower  the  tube  till 
the  objective  touches  the  oil.  As  the  field  is  now  very 
dark,  open  the  diaphragm  slig-htly.  Focus  the  edg-e  of  the 
drop  with  the  fine  adjustment,  holding-  the  slide  between 
the  fing-ers  of  the  left  hand.  Carefully  study,  in  the  man- 
ner already  indicated,  the  bacteria  which  are  present  at 
the  edge,  and  in  the  different  parts  of  the  drop. 

Laboratory  work.  —  The  student  will  make  hanging-drops  of  the 
several  chromogenic  bacteria,  which  have  been  grown  on  potatoes, 
according  to  the  directions  given  in  the  following  chapter.  Too 
much  time  cannot  be  devoted  to  this  method  of  examination,  inas- 
much as  the  practice  thus  obtained  is  indispensable  to  the  easy  and 
successful  manipulation  of  the  microscope  in  the  subsequent  work. 
A  delicate  touch,  and  a  quick  perception  of  the  smallest  detail,  can 
only  be  acquired  by  repeated  effort. 

When  the  examination  of  a  specimen  of  living-  bacteria 
is  completed,  the  cover-glass  should  be  carefully  removed, 
and  placed  in  boiling  water  for  10  or  15  minutes,  or  in  mer- 
curic chloride  solution  (1  —  1000)  for  some  hours.  If,  in  re- 
moving the  cover-glass,  the  suspension  touches  the  slide, 
the  latter  should  likewise  be  at  once  disinfected. 


Staining  of  Bacteria. 


One  of  the  most  important  conditions  when  staining 

bacteria  is  to  have  perfectly'  clean  cover-glasses.      The 
10 


146  BACTERIOLOGY. 

latter  should  be  so  clean  that,  when  a  drop  of  water  is 
placed  on  any  of  them,  and  spread  over  the  surface  by 
means  of  a  platinum  wire,  it  will  remain  spread  out  as  a 
thin  film.  If  it  gathers  in  minute  droplets,  which  follow 
the  wire  and  refuse  to  spread  out,  the  cover-glass  is  not 
clean,  and  is  not  suitable  in  that  condition  for  staining  pur- 
poses. The  thin  layer  of  fatty  matter  must  be  removed, 
and  this  is  often  impossible  when  the  ordinary  procedure  is 
followed.  The  method  of  obtaining  absolutely  clean  cover- 
glasses  has  been  described  on  p.  140. 

Anilin  dyes  are  employed  for  staining  bacteria.  It  is 
customary  to  speak  of  acid  and  basic  anilin  dyes.  The  acid 
dyes  do  not  stain  bacteria,  or  but  feebly,  and  are,  there- 
fore, useful  as  contrast  colors.  Eosin  belongs  to  this  group, 
and  its  value  will  be  seen  in  the  double  staining  of  patho- 
genic bacteria.  The  basic  anilin  dyes  stain  the  nuclei  of 
cells  and  bacteria  equally  well.  They  are,  therefore,  in- 
variably used  when  staining  bacteria.  The  basic  anilin 
dyes,  most  frequently  used,  are  fuchsin,  gentian  violet, 
methyl  violet,  and  methylene  blue.  Occasionally,  vesuvin 
or  Bismarck  brown,  is  employed. 

The  fuchsin  and  gentian  violet  are  to  be  preferred,  and 
used  for  all  ordinary  purposes.  They  stain  rapidly  and 
deeply,  and  the  preparations,  when  properly  mounted,  will 
not  appreciably  alter,  even  after  many  years.  Methylene 
blue  stains  slowly,  and  is  excellent  for  special  purposes. 
It  is  not,  however,  a  permanent  stain,  and  such  preparations 
are  likely  to  fade  after  a  short  time. 

The  dyes  mentioned  above  are  dissolved  to  complete 
-saturation,  in  absolute,  or  in  strong  alcohol.  These  con- 
centrated solutions  are  never  used  as  such,  but  serve  as  a 
stock,  from  which  the  dilute  stain  which  is  employed  can 
be  readily  prepared. 

If  the  alcoholic  solution  of  the  stain  is  diluted  too 
much,  the  dye  will  tend  to  precipitate,  and  may  yield  un- 
sightly deposits  on  the  cover-glass.  It  is  advisable,  there- 


STAINING  OF  BACTERIA.  147 

fore,  not  to  dilute  till  the  liquid  is  transparent.  If  a  cloud- 
iness forms  after  a  few  days,  the  stain  should  be  discarded 
and  a  fresh  solution  prepared.  The  best  results  are  ob- 
tained when  the  dilute  stain  contains  but  little  alcohol.  If 
the  stock  solution  is  incompletely  saturated  it  will,  on  dilu- 
tion, yield  a  transparent,  feeble  stain. 

The  dilute  anilin  stain  is  prepared  as  follows:  Some 
of  the  saturated,  alcoholic  solution  of  the  dye  is  placed  in 
a  one-ounce  bottle,  and  then  diluted  with  three  or  four 
parts  of  water.  The  bottle  should  be  provided  with  a  cork 
through  which  passes  a  glass  tube,  the  lower  end  of  which 
is  slightly  drawn  out.  This  then  serves  as  a  pipette  (Fig.  22, 
p.  150).  A  drop  of  stain,  as  it  leaves  the  pipette,  should 
be  opaque.  If  this  is  not  the  case  it  is  due,  either,  to  the  use 
of  an  unsaturated  stock  solution,  or,  to  excessive  dilution. 

Simple  staining. — Place  a  small  drop  of  water  on  a 
cover-glass  which  is  held  between  the  thumb  and  forefinger 
of  the  left  hand.  The  drop  of  water  should  not  be  much 
larger  than  a  good  sized  pin-head.  By  means  of  the  sterile 
platinum  wire,  a  portion  of  the  growth  is  transferred  to  the 
droplet  of  water.  The  latter  should  now  be  distinctly 
cloudy.  Sterilize  the  wire  again  by  heating  in  the  flame, 
and,  when  cold,  spread  out  the  droplet  of  water  over 
the  surface  of  the  cover-glass.  The  water  evaporates 
rapidly,  if  the  drop  is  small,  and  leaves  a  perfectly  even 
residue  distributed  over  the  entire  surface.  If  the  drop  is 
large,  it  will  dry  slowly  and  will  leave  unsightly  "shore 
lines." 

The  evaporation  of  the  water  may  be  hastened  by  waving-  the 
cover-glass,  to  and  fro,  a  few  inches  above  a  flame.  Care  must  be 
taken  not  to  transfer  too  much  material  to  the  drop  of  water,  for  in 
such  a  case  the  cover-glass  on  subsequent  staining  will  be  found  to  be 
one  mass  of  bacteria. 

Instead  of  making-  the  suspension  on  the  cover-glass  as  indicated 
above,  the  beginner  will  find  it  better  to  proceed  as  follows:  Place  one 


148  BACTERIOLOGY. 

or  two  large  drops  of  water  on  a  slide  and  touch  the  wire,  laden  with 
the  growth,  into  this  water  until  a  cloudy  suspension  results.  Touch 
a  straight  sterile  wire  to  this  liquid  and  transfer  the  droplet  that 
adheres  to  a  cover-glass.  The  thin  film  dries  almost  immediately 
and  is  not  likely  to  contain  too  many  bacteria.  Any  number  of 
preparations  can  be  made  from  the  original  suspension. 

A  sliminess  of  the  liquid,  and  a  tendency  of  the  bacteria  to 
clump,  can  be  overcome  by  gently  heating  the  suspension  over  a  flame. 

As  soon  as  the  water  has  evaporated  the  material  is 
"fixed"  to  the  cover-glass  by  means  of  heat.  For  this  pur- 
pose the  cover-glass  is  taken  up  in  the  forceps,  specimen 
side  up,  and  is  touched,  from  above  downward,  once  or  twice, 
to  a  Bunsen,  or  alcohol  flame.  This  little  operation  re- 
quires judgment  and  care.  The  cover-glass  must  become 
heated  sufficiently  to  fix  the  specimen,  so  that  it  will  not 
wash  off  when  subsequently  treated  with  the  dye  and  with 
water.  On  the  other  hand,  too  much  heating'  will  destroy 
the  organism  and  render  it  incapable  of  taking1  up  the  dye. 
The  best  rule  to  follow  is  to  touch  the  cover-glass,  once  or 
twice,  to  the  flame  and  then  to  bring  it  immediately  into 
contact  with  the  end  of  the  finger.  If  the  cover-glass  is  so 
hot  that  the  finger  must  be  withdrawn  at  once,  it  indicates 
that  the  specimen  is  fixed  and  that  it  is  not  necessary  to 
heat  any  more. 

The  cover-glass,  with  the  specimen  side  still  turned  up, 
is  held  in  the  forceps  and  a  drop  or  two  of  water  is  placed 
on  the  specimen.  Then,  two  or  three  drops  of  the  dilute 
gentian  violet,  or  fuchsin  are  added,  and  allowed  to  act  for 
for  one-quarter  to  one-half  minute.  The  cover-glass  is  then 
washed  perfectly  clean  of  the  dye,  by  being  held  under  a 
tap,  or  by  rinsing  in  one  or  two  glasses  of  water.  It  is 
then  touched  edgewise  to  a  piece  of  filter  or  blotting  paper 
in  order  to  drain  off  the  excess  of  water,  and  is  then  placed 
on  the  paper  with  the  specimen  side  turned  up.  By  gentle 
rotation  of  the  cover-glass,  the  lower  surface  becomes  per- 
fectly dry.  This  can  be  easily  done  if  the  paper  is  sup- 
ported on  the  tip  of  the  index  finger. 


•STAINING  OF  BACTERIA.  149 

The  cover-glass  is  now  inverted,  with  the  moist  speci- 
men side  downward,  on  a  clean  glass-slide.  Or,  the  latter 
may  be  brought  down  over  the  cover-glass  till  it  touches. 
The  specimen  should  be  examined  to  see  if  the  upper  sur- 
face of  the  cover-glass  is  perfectly  dry.  Furthermore,  just 
sufficient  water  should  be  present  between  the  cover-glass 
and  slide  to  keep  the  former  in  position.  The  .specimen 
should  never  be  examined  in  the  dry  condition.  If  there 
is  insufficient  water,  a  drop  should  be  placed  near  the 
edge  of  the  cover-glass.  Too  much  water  must  be  avoided, 
since  it  would  float  the  cover-glass,  in  which  case  the 
examination  with  the  oil  immersion  objective  is  quite  im- 
possible. Excess  of  water  can  be  drained  off  by  touching 
the  edge  of  the  cover-glass  with  a  piece  of  filter-paper. 
The  water  under  the  cover-glass  should  be  colorless,  which 
will  be  the  case  if  the  specimen  was  properly  washed. 

The  slide,  thus  prepared,  is  placed  on  the  stage  of  the 
microscope  and  examined  first  with  the  No.  7  objective  and 
subsequently  with  the  iVth  homogeneous  oil  immersion 
objective,  in  the  same  way  as  was  done  when  examining 
hanging-drops. 

A  good  specimen  should  show  the  bacteria  deeply 
stained,  not  in  masses,  but  separated  from  each  other,  and 
evenly  distributed  over  the  entire  cover-glass.  If  the 
stained  bacteria  are  seen  to  move  about,  it  is  due  to  insuf- 
ficient fixing*  in  the  flame. 

Failure  to  take  the  stain  properly  may  be  due  to  over- 
heating while  fixing  the  specimen,  or  to  a  weak  dye,  or  to  a 
too  short  exposure  to  the  dye.  By  repeated  trials,  the  ex- 
act conditions  necessary  can  be  ascertained,  and  then  fol- 
lowed without  any  difficulty. 

In  the  method  as  described  above,  the  dye  is  not  added 
direct  to  the  cover-glass,  but  to  a  drop  of  water  which  is 
first  placed  upon  it.  This  little  deviation  from  the  process, 
as  ordinarily  described,  serves  to  prevent  over-staining  and 
deposition  of  coloring  matter. 


150 


BACTERIOLOGY. 


The  color  can  be  forced  into  the  specimen,  thus  making- 
it  stand  out  more  sharply  than  would  otherwise  be  the  case, 
by  holding'  the  glass-slip,  covered  with  the  dye,  over  a  low 
Bunsen,  or  alcohol  flame  till  vapors  begin  to  rise.  The 
specimen  is  then  washed  as  before. 

Instead  of  heating-  the  dye  on  the  cover-glass,  it  may  be 
heated  before  use.  For  this  purpose  the  bottle  of  dye  is 
placed  on  an  iron  plate,  and  this  is  heated  by  a  low  flame. 
Fig".  22  shows  such  an  iron  plate,  as  used  in  the  author's 
laboratory,  slipped  under  the  flang-e  of  the  ordinary  water- 
bath.  This  plate  is  3.5  mm.  thick,  7.5  cm.  wide,  and  15 
cm.  long. 


FIG,  22.    Water-bath  with  iron  plate  for  heating  dyes,  cover- 
glassses,  etc. 

When  it  is  desirable  to  preserve  a  stained  specimen,  this 
should  be  floated  off  the  slide  by  the  addition  of  a  drop,  or 
two,  of  water  to  the  edge  of  the  cover-glass.  If  cedar  oil 
has  been  used  it  should  be  carefully  removed  by  rotating 
the  cover-glass  on  a  piece  of  filter  paper.  The  preparation 
should  then  be  placed,  specimen  side  turned  up,  under  a 
watch-glass  until  perfectly  dry.  Or,  the  drying  may  be 


STAINING  OF  BACTERIA.  151 

hastened  by  waving  the  cover-glass  over  a  name.  A  drop 
of  Canada  balsam  is  then  placed  on  the  center  of  a  clean 
slide  and  the  thoroughly  dried  cover-glass  is  inverted  and 
brought  down  on  the  balsam,  with  the  specimen  side  turned 
down.  On  gentle  pressure,  aided  by  slight  heating  of  the 
slide,  if  need  be,  the  balsam  will  spread  out  evenly  under 
the  cover-glass.  If  the  cover-glass  has  not  been  properly 
dried,  the  specimen  will  be  hazy,  and  may  even  show  drop- 
lets of  water.  In  either  case,  the  stain  will  rapidly  fade. 

The   entire   process  of  simple  staining  can  be  briefly 
summarized  as  follows: 

Clean  cover-glass. 

Spread  specimen. 

Dry  in  air. 

Fix  in  flame. 

Add  drop  of  water. 

Add  dilute  dye  (i  -  -J-  minute). 

Wash  in  water. 

Examine  in  water. 

Dry  in  air. 

Mount  in  balsam. 

Laboratory  work. — The  student  should  practice  staining"  cover-glass 
preparations  made  from  the  different  potato  cultures  (p.  170).  The  sev- 
eral dyes  mentioned  above  should  be  employed.  An  early  mastery  of 
this  truly  simple  process  of  staining-  will  save  much  time  during  the 
subsequent  work.  Preparations  should  also  be  made  from  the  white 
matter  on  the  teeth,  near  the  edge  of  the  gums,  and  these  should  be 
examined  for  micrococci  and  bacilli,  and  especially  for"  comma 
bacilli,"  spirals,  and  leptothrix  threads. 


.CHAPTER  VII. 
GELATIN  AND   POTATO    MEDIA.— CULTIVATION    OF    BACTERIA. 

The  study  of  bacteria  is  not  limited  to  the  mere  obser- 
vation of  these  organisms  under  the  microscope.  Indeed, 
as  long1  as  this  was  of  necessity  the  case  bacteriology  was 
far  from  being-  an  exact  science.  A  drop  of  blood  in  an- 
thrax, or  a  drop  of  a  putrid  liquid,  may  teem  with  bacteria. 
If  the  examination  is  limited  to  the  use  of  a  microscope,  no 
positive  conclusion  can  be  formulated  as  to  the  relation  of 
these  organisms  to  the  condition  in  which  they  occur.  An 
exact  knowledge  of  their  function  was  possible,  when  meth- 
ods were  discovered  for  artificially  cultivating  these  organ- 
isms. 

The  first  requirement,  in  order  to  accomplish  this  ob- 
ject, is  the  preparation  of  suitable,  sterile,  nutrient  media. 
The  soil  on  which  the  bacteria  in  question  are  to  be  planted, 
must  be  absolutely  free  from  other  forms  of  bacterial  life. 
Otherwise,  foreign  adventitious  organisms  may  develop  and 
outgrow  the  particular  form  under  observation.  The  use 
of  sterile  infusions,  or  nutrient  liquid  media  was  introduced 
at  an  early  date  by  Pasteur. 

When  the  material  mentioned  above  is  planted  in  a  ster- 
ile, liquid  medium,  it  is  evident  that  each  of  the  different 
kind  of  bacteria  that  may  be  present  will  multiply.  The 
result  is  a  "mixed  culture."  With  liquid  media,  it  is  ex- 
tremely difficult  to  separate  the  several  bacteria  that  may 
be  present  in  a  mixture.  The  introduction  of  nutrient  gela- 
tin by  Koch,  in  1881,  rendered  this  a  relatively  easy  task. 
The  remarkable  progress  in  bacteriology  during  the  past 
two  decades,  is  largely  the  result  of  this  simple  transforma- 


I 


GELATIN  AND  POTATO  MEDIA.  153 

tion  of  a  beef-tea  into  a  transparent,  solid,  and  easily  lique- 
fiable  medium. 


Preparation  of  Nutrient  Gelatin. 

Place  500  g.  of  chopped,  lean  beef  in  an  enamelled  jar, 
such  as  is  shown  in  Fig.  23.     This  has  a  capacity  of 
liters.      Now  add  1000  c.c.  of  tap- water  and 
stir  thoroughly.     Immerse  the  jar  in  a  water- 
bath,  and  warm  gently,  till  the  temperature  of 
the  meat  suspension  reaches  55-60°. l     Main- 
tain this  temperature  .for  f-1   hour,  stirring 
frequently.    The  soluble  constituents  are  thus 
brought  into  solution.    Special  care  should  be 
taken  not  to   allow  the   temperature  to  rise 
above  60°,  inasmuch  as  the  albuminous  sub- 
stances would   then   coagulate.     By  keeping  mfki£g2medm.r  for 
these  in  solution  at  this  stage,  they   will  sub- 
sequently assist  in  clarifying  the  final  product,  and  hence, 
the  addition  of  a  white  of  an  egg  will  be  unnecessary. 

When  the  digestion  is  completed,  strain  the  liquid 
through  muslin  and  thoroughly  squeeze  the  residue.  The 
filtrate  is  dark  red  in  appearance  and  should  measure  1000 
c.c.  [If  this  is  not  the  case,  add  water  to  make  up  this 
volume.  To  1000  c.c.  of  the  filtrate  returned  to  the  clean 
jar,  now  add 

100  g.  of  gelatin, 

10  g.  of  dry  pepton  (Witte's), 

5  g.  of  common  salt, 

and  warm  at  60°,  in  the  water-bath,  with  constant  stirring, 
till  the  gelatin  has  completely  dissolved.  The  next  step  is 
to  render  the  liquid  slightly,  but  distinctly  alkaline. 

1  A  thermo-r  emulator  may  be  used  to  maintain  a  constant  temper- 
ature in  the  water-bath  (see  Chapter  IX,  also  Fig-.  75). 


154  BACTERIOLOGY. 

The  neutralization  is  usually  accomplished  by  the  cautious  addi- 
tion of  a  saturated  solution  of  sodium  carbonate.  After  each  addi- 
tion of  1  to  2  c.c.  of  the  alkali,  the  liquid  is  well  stirred,  and  a  drop  is 
taken  out  by  means  of  a  giass  rod  and  touched  to  a  blue  litmus  paper. 
If  the  reaction  is  acid,  this  paper  will  turn  red.  The  addition  of  al- 
kali is  continued  until  the  blue  paper  retains  its  color,  and  the  red 
litmus  paper  turns  slightly  blue. 

In  the  hands  of  the  beginner,  this  procedure  not  infrequently 
fails,  because  of  the  difficulty  in  judging-  the  end-reaction.  Moreover, 
even  the  practised  eye  cannot  establish  the  same  degree  of  alkalin- 
ity in  two  separate  preparations.  For  these  reasons,  some  workers 
prefer  to  titrate  the  solution  with  an  alkali  of  known  strength,  using 
phenol-phthalein  as  an  indicator.  The  latter  is  a  most  delicate  indi- 
cator when  mere  aqueous  solutions  of  acid  and  alkali  are  to  be  tested. 
In  the  presence  of  organic  matter,  ammonium  salts  and  carbonic  acid 
it  ceases  to  be  a  sharp  indicator.  Moreover,  the  neutral  point,  as  ob- 
tained with  phenol-phthalein,  does  not  correspond  with  the  neutral 
point  obtained  with  litmus.  Indeed,  gelatin  neutralized  thus,  is  in- 
tensely alkaline  to  litmus.  Consequently,  it  has  been  found  necessary 
to  deduct  20  or  25  c.c.  from  the  total  amount  of  alkali  necessary  to 
neutralize  a  liter  of  the  medium.  The  amount  thus  subtracted  is  so 
arbitrary  that  the  resulting  reaction  cannot  be  duplicated,  except  ap- 
proximately, in  another  batch  of  the  same  or  of  other  media. 

The  following1  method  determines,  with  reference  to  lit- 
mus, the  neutral  point  of  any  medium,  whether  gelatin,  bouil- 
lon, or  agar  to  a  degree  of  exactitude  that  leaves  nothing  to 
be  desired.  The  beginner,  with  no  previous  knowledge  of 
quantitative  analysis,  can  impart  any  desired  degree  of  al- 
kalinity (or  acidity)  to  a  given  medium. 

In  order  to  neutralize  the  gelatin  solution,  it  is  neces- 
sary to  prepare  two  solutions  of  sodium  hydrate. 

1. — One  that  will  contain  40  g.  of  this  base  in  one  liter. 
This  is  known  as  normal  (N)  sodium  hydrate.  To  prepare 
this  solution  so  that  it  will  have  exactly  this  strength 
requires  some  experience  in  methods  of  quantitative 
analysis.1 

1  For  the  preparation  of  exact  normal  and  deci-normal  solutions 
the  student  is  referred  to  the  author's  Laboratory  Work  in  Physiolog- 
ical Chemistry,  2nd  ed.,  pp.  220-239. 


GELATIN   AND    POTATO  MEDIA. 


155 


For  practical  purposes  it  is  sufficient  to  dissolve  40  g.  of 
sodium  hydrate  in  distilled  water,  and  to  dilute  this  solution 
to  one  liter.  This  will  give  an  approximately  normal  solu- 
tion. 

2. — One  that  will  contain 
4  g.  of  this  base  in  one  liter 
of  water.  This  is  known  as  a 
deci-normal  (ft)  sodium  hydrate 
solution.  It  is  prepared  by 
taking  100  c.c.  of  the  normal 
solution,  and  diluting-  this 
with  distilled  water  to  one 
liter.  It  is  evident  that  this 
solution  has  one-tenth  the 
strength  of  the  former.  These 
two  solutions  are  placed  in 
the  apparatus  shown  in  Fig. 
24.  This  consists  of  two 
burettes,  each  of  50  c.c.  capa- 
city, graduated  in  T<T  c.c.,  and 
connected  with  bottles  or 
flasks  which  contain  the  nor- 
mal and  decinormal  solutions, 

reSDCCtivelv  FIG.  24.    Burettes  for  titrating  nutrient 

media,    a— Connected  with  stock  of  ft  NaOH 

Titration  Of  the  Gtelatin. — Bv  solution.       £— Connected     with     stock     of 

J   N  NaOH  solution  (F.G.N.). 

means  of  a  pipette   measure 

out  10  c.c.  of  the  gelatin  solution  into  each  of  four  test- 
tubes,  and  label  these  1,  2,  3,  and  4. 

To  tube  1  add  2.5  c.c.  of  the  ft  NaOH  solution. 
To  tube  2  add  2.8  c.c.  of  the  ft  NaOH  solution. 
To  tube  3  add  3.2  c.c.  of  the  &  NaOH  solution. 
To  tube  4  add  3.5  c.c.  of  the  ft  NaOH  solution. 

Heat  each  tube  in  the  flame  till  the  liquid  boils,  in 
order  to  expel  carbonic  acid,  and  to  precipitate  phosphates 
and  albumin.  Then,  drop  into  each  tube  a  strip  of  blue 


156  BACTERIOLOGY. 

and  one  of  red  litmus  paper.  These  should  be  immersed  in 
the  liquid.  Again,  heat  to  boiling  and  set  aside  for  one 
minute.  By  means  of  a  glass  rod  draw  out  the  papers,  side 
by  side,  upon  the  wall  of  each  tube  and,  when  cold,  compare 
the  colors  of  the  papers,  by  holding  the  tubes  before  a  win- 
dow, over  a  white  surface.  What  was  originally  the  blue 
paper  in  tube  1,  will  probably  have  a  slight  red  color. 
This  shows  that  2.5  c.c.  of  ft  NaOH  is  not  sufficient  to  neu- 
tralize 10  c.c.  of  the  gelatin  solution.  On  the  other  hand, 
in  tube  4,  both  papers  are  blue,  which  indicates  that  3. 5  c.c. 
of  the  alkali  is  more  than  is  necessary  to  neutralize  10  c.c. 
of  the  liquid.  The  neutral  point,  that  is,  where  the  blue 
and  red  paper  retain  their  color  side  by  side,  lies  therefore 
between  these  two  extremes.  Tube  2,  apparently,  shows  a 
slight  acid  reaction,  whereas  tube  3,  is  faintly  alkaline. 
The  neutral  point  lies,  therefore,  between  2.8  and  3.2;  it 
probably  is  about  3.0.  Hence,  measure  out  into  a  test-tube 
10  c.c.  of  the  gelatin,  and  add  3.0  c.c.  of  the  ft  NaOH. 
Then  boil  and  add  litmus  paper,  and  examine  as  above.  In 
this  way  it  is  possible  to  determine  the  neutral  point  to 
within  0.1  or  0.2  of  a  c.c.,  and  this  corresponds  to  a 
probable  error  of  1  or  2  c.c.  of  N  NaOH  per  liter  of 
gelatin. 

The  above  experiment  has  shown  that  10  c.c.  of  the 
gelatin  requires  3.0  c.c.  of  ft  NaOH  for  neutralization.  In 
order  to  ascertain  the  amount  of  alkali  necessary  for  the 
neutralization  of  all  of  the  remaining  gelatin,  the  latter 
must  be  measured  in  a  cylindrical  graduate.  The  amount 
left  corresponds,  for  example,  to  950  c.c.  The  amount  of 
&  alkali  necessary  to  neutralize  this  quantity  is  ascertained 
from  the  following  proportion: 

10  :  3  :  :  950  :  x         x  =  285. 

That  is  to  say,  in  order  to  neutralize  the  950  c.c.  of  ge- 
latin, it  would  be  necessary  to  add  285  c.c.  of  ft  NaOH. 
Since  this  corresponds  to  28.5  c.c.  of  N  NaOH,  the  latter  is 


GELATIN   AND  POTATO  MEDIA.  157 

added  in  preference  to  the  large  volume  of  &  alkali,  which 
would    unnecessarily    dilute    the    liquid. 

Bacteria  grow  best  on  slightly  alkaline  media.  It  is, 
therefore,  desirable  to  add  an  amount  of  alkali,  over  and 
above  that  necessary  for  mere  neutralization.  An  excess 
of  10  c.c.  of  N  NaOH  per  liter  imparts  a  desirable  alkalin- 
ity. Hence,  to  the  950  c.c.  of  gelatin  there  will  be  added 
28.5  c.c.  of  N  NaOH  for  neutralization,  and  9. 5  c.c.  for  alka- 
linity—a total  of  38  c.c.  of  N  NaOH. 

After  the  addition  of  the  necessary  amount  of  alkali  to 
the  remaining  gelatin  in  the  jar,  the  latter  is  then  placed  in 
the  water-bath.  The  water  is  now  raised  to  boiling,  and  is 
maintained  at  this  point  for  ^-1  hour.  The  albuminous 
substances,  which  are  present  in  the  meat  [extract,  co- 
agulate in  flakes,  and  clarify  the  liquid  so  that  on  subse- 
quent nitration  it  will  be  perfectly  clear. 

The  liquid  is  then  filtered  through  a  plaited  filter. 
These  may  be  obtained  ready  made.  Schleicher  and 
Schiill's  No.  580  is  particularly  well  adapted  for  filtering 
gelatin.  It  is  advisable  to  pass  about  200  c.c.  of  boiling 
water  through  the  filter,  before  beginning  the  filtration.  If 
the  first  portion  of  the  filtrate  is  cloudy,  it  should  be 
returned  to  the  filter.  The  filtrate  should  be  (1)  perfectly 
clear;  (2)  should  be  neutral  or  slightly  alkaline  in  reaction; 
(3)  should  not  become  cloudy  or  coagulate  when  boiled  in  a 
test-tube  for  1  to  2  minutes;  (4)  should  solidify  when  cooled. 

If  the  filtrate  is  cloudy  and  strongly  alkaline,  correct 
the  reaction  by  the  addition  of  a  few  drops  of  dilute  acetic 
acid.  If  it  becomes  cloudy,  or  coagulates  when  warmed, 
continue  heating  in  the  water-bath.  A  cloudiness  can  be 
cleared  up  by  the  addition  of  a  white  of  an  egg  dissolved 
in  a  little  water.  The  commercial,  dry  egg-albumin  is  con- 
venient for  this  purpose.  In  either  case,  the  albumin  solu- 
tion should  not  be  added  to  the  boiling  solution,  but  this 
should  first  be  cooled  to  about  60°.  The  temperature  should 


158  BACTERIOLOGY. 

then  be  raised  slowly  to  the  boiling  point,  and  the  liquid 
stirred  constantly.  By  following  the  directions  given  in 
the  preparation  of  the  meat  extract  it  will  not  be  neces- 
sary to  resort  to  clarification  with  egg-albumin.  If  the 
gelatin  fails  to  solidify  on  cooling  it  is  because  it  has  been 
heated  too  long.  In  that  case  more  gelatin  should  be  added 
(50  g.),  the  liquid  then  neutralized  and  treated  as  before. 

If  the  filtered  gelatin  answers  the  above  requirements 
it  is  ready  to  be  "tubed",  that  is,  filled  into  sterile  tubes. 
This  should  be  done  by  means  of  a  small  glass  funnel !  and 
the  tubes  should  be  filled  to  a  depth  of  1^  inches.  The 
large  tubes  will  then  contain  about  8  c.c.  of  gelatin.  The 
utmost  care  should  be  taken  to  avoid  touching  the  neck  of 
the  tube  with  the  gelatin,  since  otherwise  the  cotton  will 
adhere  to  the  tube.  The  tubes,  filled  with  gelatin,  are 
placed  in  the'copper  sterilizing  pail  (Fig.  26,  p.  164). 

The  tubes  employed  are  15  or  16  mm.  in  diameter  and 
150  mm.  long.  Although  the  tubes  may  be  new  and  ap- 
parently clean,  they  should  be  invariably  washed  with  hot 
water,  preferably  slightly  acidulated,  in  order  to  remove  any 
alkali  that  may  remain  from  the  manufacture.  They  should 
then  be  rinsed  in  cold  water,  and  allowed  to  drain  till  dry. 
The  tubes  are  then  plugged  with  cotton.  The  simplest  way 
of  doing  this  is  to  place  a  piece  of  cotton,  1^  to  2  inches 
square,  on  the  mouth  of  the  tube  and  then  pushing  down 
the  middle  by  means  of  a  narrow  glass  tube,  rod,  or  match- 
stick.  The  best  and  most  solid  plugs  are  obtained,  by  fold- 
ing over  a  piece  of  cotton  into  thirds  and  the  rolling  up 
from  the  end.  A  firm,  cylindrical  plug  is  thus  obtained 
without  any  frayed  out  border.  The  plug  should  be  1  to  1| 
inches  long  and  should  project  out  of  the  tube  as  little  as 
possible.  The  nutrient  medium  can  be  filled  into  the  tubes 
which  have  not  been  sterilized.  The  subsequent  exposure 
to  steam  will  bring  about  sterilization  of  the  walls  of  the 

1  The  globe  funnel  shown  in  Fig-.  72  can  be  used  for  the  rapid  filling 
of  tubes. 


GELATIN   AND  POTATO   MEDIA.  159 

tube,  of  the  plug",  and  of  the  contents.  It  is  customary, 
however,  to  subject  the  tubes,  before  they  are  filled  with 
culture  media,  to  sterilization  by  means  of  dry-heat.  It  is 
desirable  to  do  this  owing-  to  the  presence  of  higiily  resist- 
ant spores  in  the  cotton,  or  in  the  tubes.  The  tubes  are 
placed  erect  in  baskets  made  of  heavy  wire  gauze. 

Dry-heat  Sterilization. 

By  sterilization  is  meant  the  total  destruction  of  all 
living-  forms,  in  or  about  the  object  exposed  to  the  process. 
Heat  is  the  most  effective  agent  in  securing-  this  condition. 
It  may  be  used  as  dry-heat  or  as  moist-heat.  Each  of 
these  has  its  special  advantages.  Dry-heat  is  used  only, 
for  the  sterilization  of  glass  and  metal  instruments  or 
articles.  Owing  to  the  high  temperature  employed, 
150-170°,  culture  media  and  other  organic  substances  cannot 
be  thus  treated,  since  charring  would  promptly  result.  For 
such  substances  moist,  steam-heat  is  usually  employed. 

The  dry-heat  sterilizer,  or  oven,  is  made  of  sheet-iron  and  is 
double-walled,  thus  permitting-  the  circulation  of  hot  air  around  the 
inner  compartment.  The  heat  is  applied  by  means  of  a  good  Bunsen 
burner.  As  usually  constructed,  the  heat  is  directed  against  the 
bottom  of  the  inner  compartment.  In  time,  this  bottom  burns  out 
and  permits  the  flame  to  enter.  The  apparatus,  in  that  case,  cannot 
be  readily  repaired.  This  defect  in  construction  can  be  readily 
obviated  by  attaching-,  to  the  bottom,  a  loose  plate,  which  is  held  in 
position  by  a  pin,  or  nut.  This  plate,  when  burned  out,  can  be 
replaced  at  a  merely  nominal  expense.  The  sterilizer  can  be  still 
further  improved  by  placing-  the  two  opening's,  which  are  on  top,  in 
the  rear  corners.  Through  each  of  these  openings  is  passed  a  brass 
tube,  freely  perforated  on  all  sides  (see  Fig.  25).  These  tubes  are 
intended  to  receive  a  thermometer  and  a  thermo-regulator,  respect- 
ively. These  instruments  are  thus  protected  against  accidental 
injury  and  do  not  interfere  with  the  placing  in,  or  the  taking  out  of 
baskets  and  other  apparatus.  Pig.  25  shows  a  dry-heat  sterilizer 
which,  however,  is  twice  the  usual  length.  The  thermo-regulator  in- 


160 


BACTERIOLOGY. 


dicated  is  of  the  author's  own  construction  (see  Fig.  37).     As  a  rule,, 
a  regulator  is  not  necessary  when  using  the  dry-heat  sterilizer. 


FIG.  25.    Dry-heat  sterilizer,  double  width.    In  the  corners,  perforated  brass 
tubes  to  protect  the  thermometer  and  the  thermoregulator. 

The  time  necessary  to  effect  sterilization  in  the  dry- 
heat  oven  will,  of  course,  depend  upon  the  temperature. 
The  ordinary  laboratory  articles  are  quickly  sterilized  at 
150°.  When  the  thermometer  indicates  150°,  the  time 
should  be  noted,  and  this  temperature  should  then  be  main- 
tained for  y?,  to  y^  of  an  hour.  Glass  tubes  and  dishes 
are,  therefore,  to  be  sterilized  in  the  dry-heat  oven  at  a 
temperature  of  150-160°  for  ^-^  of  an  hour.  A  tem- 
perature of  170°,  maintained  for  15  minutes,  will  be  suffi- 
cient to  effect  sterilization.  Another  procedure  is  to  allow 
the  temperature  to  rise  till  it  reaches  195°,  when  the  light 
is  turned  out.  The  door  of  the  oven  remains  closed  until 
the  temperature  falls  below  100°.  The  cotton  plug's  in  the 


GELATIN   AND   POTATO  MEDIA.  161 

tubes,  which  have  been  subjected  to  sterilization,  should 
show  a  light  yellow  tinge. 

Steam  Sterilization. 

When  the  gelatin  is  filled  into  tubes,  these  must  be 
again  subjected  to  sterilization.  If  this  is  not  done,  or 
done  improperly,  bacteria  will  promptly  develop  in  the 
gelatin  and  render  it  worthless.  Such  instances  of  '  *  spon- 
taneous generation"  are  the  result  of  insufficient  exposure 
to  steam-heat.  As  indicated  above,  cultural  media,  such  as 
gelatin,  are  never  sterilized  in  the  dry-heat  oven.  The  de- 
struction of  all  the  living  forms  that  may  be  present,  is 
accomplished  by  the  aid  of  moist  heat. 

It  will  be  remembered  that  bacteria  may  exist  in  two 
stages — the  actively  vegetating  or  growing  condition,  and 
the  resting  or  spore  condition.  When  in  the  vegetating 
state,  these  organisms,  as  a  rule,  are  readily  destroyed  by 
moist  heat.  A  temperature  of  65-70°  may  do  this  in  10 
or  15  minutes,  whereas  steam-heat  (100°)  maybe  said  to  de- 
stroy the  growing  cell  instantaneously.  On  the  other  hand, 
the  spore,  or  seed,  is  exceedingly  resistant  to  destruction. 
Moist  heat  of  65-70°  has  little  or  no  action,  and,  even 
steam-heat  may  require  some  time.  In  order  to  sterilize 
nutrient  media  it  is  customary,  therefore,  to  employ  steam- 
heat.  They  may  be  sterilized  by  one  prolonged  exposure 
to  steam  at  100°.  Thus,  the  gelatin  tubes  might  be  steril- 
ized by  steaming  for  one  hour.  This  procedure,  indeed,  is 
sometimes  resorted  to  when  it  is  desired  to  employ  the  me- 
dium on  the  same  day  on  which  it  is  prepared. 

Prolonged  steaming  tends  to  alter  a  medium;  it  may 
render  the  latter  acid,  and,  in  the  case  of  gelatin,  may  soften 
this  to  such  an  extent  that  it  will  not  congeal  on  subsequent 
cooling.  By  taking  advantage  of  the  difference  in  the  resist- 
ance offered  by  the  vegetating  and  spore  forms,  it  is  possible 

11 


162  BACTERIOLOGY. 

to  sterilize  without  materially  altering*  the  physical  or  chem- 
ical character  of  the  medium.  An  exposure  of  15  minutes  to 
steam  will  surely  cause  the  destruction  of  all  the  vegetating 
organisms  that  may  be  present.  The  spores,  however,  sur- 
vive. If  the  medium  is  now  set  aside  at  the  ordinary  room 
temperature  for  24  hours,  all  or  nearly  all  of  the  spores  will 
germinate,  and  thus  give  rise  to  the  growing,  vegetating 
type,  The  highly  resistant  spore  has  transformed  itself 
into  a  feebly  resistant  organism.  An  exposure,  on  the  sec- 
ond day,  to  steam  for  15  minutes  will,  therefore,  result  in 
the  destruction  of  all  these  young  cells.  The  material  is 
set  aside  for  another  24  hour  period,  in  order  to  give  any 
remaining  spores  an  opportunity  to  germinate.  A  third  ex- 
posure to  steam  for  15  minutes,  on  the  third  day,  will  ren- 
der the  medium  sterile. 

This  method  is  known  as  fractional  sterilization,  and  is 
the  one  commonly  employed  in  rendering  cultural  media 
sterile.  The  exposures  of  15  minutes  each,  as  just  described, 
are  sufficient  in  the  case  of  gelatin.  When  the  medium  is  in 
bulk,  or  if,  as  in  the  case  of  agar,  it  does  not  melt  readily, 
it  will  be  necessary  to  lengthen  the  exposure  to  at  least  30 
minutes  on  each  day.  The  gelatin  tubes  are  to  be  exposed 
to  steam  for  15  minutes,  as  soon  as  the  tubes  are  filled. 
Then  on  the  second  and  on  the  third  day,  this  exposure  to 
steam  for  a  like  interval  is  repeated.  Nutrient  gelatin  is 
sterilized,  fractionally,  by  exposure  to  steam-heat  for  15  minutes 
on  each  of  three  consecutive  days. 

Sometimes,  bacteria  develop  in  tubes  which  have  apparently 
been  subjected  to  proper  sterilization.  Spores,  if  clumped  together 
in  a  mass,  will  not  all  develop  at  the  same  time.  Moreover,  the 
spores  in  the  interior  of  such  a  mass  will  resist  the  action  of  heat 
for  a  considerable  length  of  time.  Thus,  if  a  growth  of  the  potato 
bacillus,  rich  in  spores,  be  gathered  into  a  little  mass,  the  size  of  a 
grain  of  wheat,  and  placed  in  a  tube  of  bouillon,  it  can  be  exposed  to 
active  steam  for  8  or  10  hours,  or  even  long-er,  without  destroying 
all  the  organisms  present.  The  same  material  stirred  up  so  as  to 


I 


GELATIN  AND  POTATO  MEDIA.  163 

form  a  very  fine  suspension,  can  be  sterilized  in  two  to  three  hours. 
Delay  in  the  germination  of  spores,  because  of  low  temperature,  or 
because  of  shelter,  may  give  rise  to  a  contamination  of  the  medium* 

In  very  warm  weather,  as  in  mid-summer,  failure  to  obtain  per- 
fect sterilization  may  be  due  to  another  cause.  The  spores  that  may 
be  present,  after  the  first  heating-,  will  g-erminate  but  the  young-  cells, 
owing-  to  the  prevailing-  hig-h  temperature,  multiply  rapidly  and 
eventually  form  new  spores.  Consequently,  there  may  be  as  many 
spores  present  after  the  second  heat,  as  after  the  first.  Under  these 
conditions,  the  medium  can  be  steamed  on  each  of  six  or  eig-ht  days, 
and  yet  not  become  sterile.  Sterilization,  however,  can  be  accom- 
plished in  such  a  case  by  steaming  at  intervals  of  12  to  15  hours,  or  by 
keeping-  the  material  at  a  low  temperature  (18-20°). 

Failure  to  secure  sterilization  may,  in  exceptional  cases,  as 
pointed  out  by  Smith,  be  due  to  the  presence  of  spores  of  anaerobic 
bacteria.  Such  spores  are  unable  to  g-erminate  in  the  presence  of 
air,  and  consequently  may  survive  fractional  sterilization.  The  me- 
dium will  be  apparently  sterile,  and  it  is  only  when  aerobic  bacteria 
are  planted,  that  the  spores  of  the  anaerobe  are  enabled  to  g-er- 
minate. 

Some  substances  cannot  be  heated  to  100°  without  profound 
chang-e.  Blood-serum,  for  instance,  will  coag-ulate  when  heated 
above  70°.  The  principle  of  fractional  sterilization,  however,  can 
be  applied  to  serum.  Since  the  temperature  cannot  exceed  even  60°, 
it  must  be  allowed  to  act  for  at  least  one  hour,  on  each  day.  This 
temperature  is  sufficient  to  kill  most  of  the  veg-etating-  forms.  The 
spore  resists  perfectly,  and  it  is  only  after  g-ermination  that  the 
org-anism  can  be  destroyed. 

The  steam  sterilizer. — The  apparatus  usually  employed 
in  Germany,  is  known  as  the  Koch  steam  sterilizer.  It  is 
essentially  a  large  cylinder,  the  lower  part  of  which  con- 
tains water.  A  grating,  at  about  one-fourth  the  distance 
from  the  bottom,  serves  to  support  the  objects  to  be  steril- 
ized. The  water  is  raised  to  boiling1  by  means  of  a  large 
burner,  and  the  steam  escapes  from  the  opening-  in  the  lid. 
Owing*  to  the  large  amount  of  water  to  be  heated,  much 
time  is  lost  in  waiting  for  the  appearance  of  active  steam. 
If,  perchance,  the  gas  pressure  is  low,  it  may  be  very 
difficult  to  obtain  any  steam.  In  this  country,  the  Arnold, 
steam  sterilizer  is  used  extensively. 


164 


BACTERIOLOGY. 


In  this  laboratory  the  Koch  sterilizer  has  been  entirely  sup- 
planted by  the  simple  sterilizer1  shown  in  Fig-.  26.  It  may  be  consid- 
£red  as  a  modification  of  the  former.  It  consists  of  the  ordinary 
Hoffmann  iron  water-bath,  which  has  an  internal  diameter  of  18  cm., 
and  a  copper  pail  (20  X  20  cm.)  which  is  provided  with  a  perforated 
bottom.  Two  perforated  rings,  on  the  inside,  allow  the  passage  of 
steam,  and  prevent  the  cotton  of  the  tubes  from  resting  ag-ainst  the 
side  of  the  pail. 


FIG.  26.    The  author's  steam  sterilizer,    a — An   ordinary  Hofmann's  iron  water-bath, 
18  cm.  in  diameter;  b — Copper  sterilizer;   c — Support  blocks. 

The  copper  pail  is  filled  with  the  gelatin  tubes,  and 
is  placed  on  the  water-bath,  in  which  the  water  should  be 
actively  boiling.  In  from  five  to  seven  minutes  steam  will 
issue  rapidly,  from  the  tube  in  the  cover,  showing  that  the 
temperature  in  the  interior  has  reached  100°.  The  steam- 
ing" is  then  continued  for  an  additional  15  minutes.  This 
process  is  now  repeated,  on  each  of  the  following  two  days. 
By  means  of  this  simple  sterilizer,  the  student  is  enabled  to 
perform  all  the  necessary  sterilization  at  his  own  table. 
Practically  no  time  is  lost  in  waiting  for  the  generation  of 

1  Centralblatt  fur  Bakteriologie,  Bd.  22,  p.  340,  1897. 


GELATIN   AND   POTATO   MEDIA. 


165 


steam,   or  for  a  sterilizer,  as  is  often  the  case,  when  the 
large  Koch  apparatus  is  employed. 

The  autoclave. — As  indicated  heretofore,  the  spore,  which 
is  the  most  resistant 
form  encountered, 
may  resist  the  action 
of  steam  even  for 
several  hours. 
Steam  escapes  from 
the  ordinary  Koch 
sterilizer  or  its  sev- 
eral modifications, 
at  the  ordinary  at- 
mospheric pressure, 
and  hence,  the  tem- 
perature never  ex- 
ceeds 100°.  If,  how- 
ever, the  steam  is 
not  allowed  to 
escape,  but  remains 
confined  in  an  appa- 
ratus, the  tempera- 
ture of  the  steam 
will  rise  with  the 
increase  in  pres- 
sure. In  this  way, 
steam  having"  a  tem- 
perature of  110°,  120° 
or  130°  can  be  readily  obtained.  The  higher  the  tempera- 
ture of  the  steam,  the  more  rapidly  will  the  resistant  spores 
be  destroyed.  Steam  at  130°,  under  pressure,  will  destroy 
instantaneously,  spores  which  are  able  to  withstand  3  or  4 
hours'  exposure  at  100°.  The  Pasteur  school  recognized, 
at  an  early  date,  the  value  of  steam  under  pressure  as  a 
means  of  effecting-  sterilization  and,  for  that  reason,  in 


FIG.  27.     Autoclave  of  Lequeux  (Wiesnegg). 


166  BACTERIOLOGY. 

France,  the  autoclave  (Pig".  27),  is  used  almost  exclu- 
sively. The  apparatus,  although  expensive,  and  requiring 
careful,  intelligent  usage,  is  by  no  means  difficult  to  handle. 
By  means  of  the  autoclave  it  is  possible  to  have  a  sterile 
medium  in  about  half  an  hour  after  this  has  been  prepared. 
No  well  equipped  laboratory  should  be  without  this  appa- 
ratus. 

A  temperature  of  120-130°,  if  allowed  to  act  for  15  min- 
utes or  longer,  may  render  the  medium  acid,  or  may  give 
rise  to  decomposition  products,  which  will  inhibit  the  sub- 
sequent growth  of  bacteria.  This  is  especially  true  of 
media  which  contain  sugar,  as  for  instance  milk.  In  either 
case  the  medium  becomes  worthless.  A  temperature  of  110° 
has  very  little  effect  on  the  media,  and,  if  allowed  to  act 
for  15  minutes  will  sterilize  the  same. 

The  following-  directions  should  be  closely  observed  when  using-  an 
autoclave: 

1.— See  that  sufficient  water  is  present. 

2. — Place  the  lid  in  position  and  fasten  tight,  ~by  hand.  The  wrench 
should  not  be  used,  if  possible. 

3. — Open  the  steam-valve,  and  lig"ht  all  the  burners.  See  that 
they  do  not  "  shoot  back." 

4. — After  the  steam  has  issued  violently  for  about  1  minute  close 
the  steam-valve.  The  steam  is  allowed  to  escape  in  order  to  expel  all 
the  air  that  is  present  in  the  autoclave. 

5.— The  pressure  now  rises,  and  when  the  g-aug-e  indicates  110°  the 
supply  of  g-as  to  the  inner  ring-  of  burners  is  shut  off,  and  that  to  the 
outer  ring-  is  turned  down  as  low  as  possible.  A  few  trials  will  enable 
one  to  turn  down  these  burners  so  that  the  temperature  of  110°  will 
continue  constant. 

6. —Maintain  a  temperature  of  110°  for  15  minutes,  then  turn  off 
all  g-as. 

7. — When  the  temperature  has  fallen  to  100°,  or  less,  the  steam- 
valve  can  be  opened,  but  not  before.  If  the  latter  is  opened  when  the 
temperature  is  above  100°,  the  sudden  release  of  pressure  will  cause 
the  liquid  in  the  tubes  or  flasks  to  boil  over.  After  the  steam-valve 
has  been  opened  the  lid  of  the  autoclave  may  be  loosened  and  re- 
moved, without  waiting  for  the  apparatus  to  completely  cool. 


GELATIN   AND    POTATO   MEDIA.  167 

8. — The  safety  valve  should  be  set  to  open  at  about  125°.  The 
noise  caused  by  the  escape  of  steam  when  this  limit  is  reached  will 
call  attention  to  the  apparatus  in  case  it  has  been  overlooked. 

The  autoclave  can  be  used,  by  leaving1  the  steam-valve  open,  for 
fractional  sterilization  at  100°. 

The  student  should  distinguish  between  steam  under 
pressure,  as  employed  above  in  an  autoclave,  and  super- 
heated steam.  A  current  of  steam,  passing1  through  a  tube, 
may  be  heated  by  a  lamp  to  130°  or  higher.  Owing  to  the 
expansion,  since  the  pressure  remains  normal,  the  steam 
will  not  have  the  destructive  action  of  that  which  has  the 
same  temperature,  but  is  under  pressure.  Indeed,  super- 
heated steam  may  be  said  to  possess  but  little  advantage 
over  a  corresponding-  degree  of  dry  heat. 

Preparation  of   Potato  Cultures. 

Select  three  sound  potatoes  and  clean  them  thoroughly, 
under  the  tap,  with  the  aid  of  a  brush.  By  means  of  a 
knife  remove  any  bad  spots,  or  depressions  that  may  exist, 
since  these  frequently  harbor  bacteria  which  are  highly  re- 
sistant to  destruction.  In  so  doing,  avoid  cutting  off  the 
skin  more  than  is  necessary.  Place  the  potatoes,  thus  pre- 
pared, in  a  solution  of  mercuric  chloride  (1  to  1,000)'  for  £  an 
hour;  then  transfer  to  a  steam  sterilizer  and  steam  for  f  of  an 
hour.  The  potatoes  should  be  well  cooked.  Allow  the 
potatoes  to  remain  in  the  pail  till  partially  cool. 

A  * '  moist  chamber "  (Fig.  28  b)  is  prepared  by  placing 
a  round  filter-paper  on  the  bottom  of  the  lower  dish,  and 
moistening  it  with  mercuric  chloride,  the  excess  of  which 
is  allowed  to  drain  off.  Three  potato  knives  are  then  ster- 
ilized. This  is  done  by  heating  the  blade  in  the  flame  till 

1  A  stock  solution  of  mercuric  chloride  is  first  prepared  by  dissolv- 
ing- 200  g.  of  the  salt  in  100  c.c.  of  concentrated  commercial  HC1. 
5  c  c.  of  this  solution  added  to  1000  c.c.  of  water  will  give  a  1:1000 
solution. 


168  BACTERIOLOGY. 

the  edge  begins  to  redden.  The  knives  are  then  set  aside 
to  cool,  with  the  edges  turned  up,  on  a  block  or  over  the 
edge  of  the  table,  care  being  taken  that  the  blade  does 
not  touch  anything. 

The  partially  cooled  potato  is  now  picked  up  with  the 
left  hand,  which  previously  has  been  dipped  in  mercuric 
chloride,  and  cut  into  halves  by  a  horizontal  section  with 
a  sterilized  knife.  Each  half  of  the  potato  is  carefully 
placed  in  the  moist  chamber,  with  the  cut  surface  turned 
upward. 

The  cut  surface  must  not  come  into  contact  with  the  fingers, 
paper  or  glass,  since  such  contact  is  very  likely  to  result  in  the  plant- 
ing- of  bacteria  on  the  potato.  The  moist  chamber  should  not  be 
allowed  to  remain  uncovered,  even  for  a  minute.  The  cut  potatoes 
remain  in  the  dish  until  perfectly  cool.  If  the  subsequent  transplan- 
tation is  carried  on  with  a  hot  knife,  or  to  a  hot  potato,  it  may  result 
in  the  killing-  of  the  organism  to  be  planted.  The  potatoes,  while 
cooling,  give  off  aqueous  vapor  which  condenses  on  the  lid  and  may, 
eventually,  drop  down  and  thus  cause  contamination.  The  con- 
densed water  should,  therefore,  be  repeatedly  removed  from  the 
cover,  by  means  of  a  piece  of  filter-paper. 

When  cold,  the  potatoes  are  ready  to  be  inoculated.  A 
small  portion,  the  size  of  a  pin-head,  of  the  bacterial 
growth  is  transferred  by  means  of  a  sterilized  and  cooled 
wire,  or  knife  to  one  of  the  above  pieces  of  a  potato,  and 
then  thoroughly  spread  over  the  surface.  Care  should  be 
taken  to  avoid  the  outer  J  inch.  The  potato  is  held  in  the 
fingers  of  the  left  hand,  which  has  been  dipped  in  mercuric 
chloride.  The  number  of  bacteria  which  is  thus  trans- 
ferred to  the  surface  of  the  potato  is  usually  so  great  that 
when  they  develop  the  entire  surface  is  covered  with  a  con- 
tinuous growth.  Such  a  growth  is  spoken  of  as  a  mass 
culture. 

The  object  in  planting  the  bacteria  on  the  potato  is  not 
so  much  to  obtain  a  mass  growth,  as  it  is  to  spread  a 
minute  portion  over  as  large  an  area  as  possible.  When, 


GELATIN   AND    POTATO  MEDIA.  169 

therefore,  as  the  next  step,  a  pin-head  portion  is  removed  by 
means  of  a  sterile  knife  from  the  surface  of  this  first  potato 
(No.  1)  it  will  contain  a  very  small  fraction  of  one  per  cent, 
of  the  total  number  of  bacteria  spread  over  the  surface  of 
that  potato.  This  relatively  small  number  of  bacteria  is 
now  spread,  or  distributed,  as  thoroughly  as  possible,  over 
the  surface  of  a  second  potato.  In  doing-  this  the  same 
precautions  are  taken  as  when  potato  No.  1  was  inoculated. 
When  the  bacteria  that  are  thus  planted  on  the  second 
potato  (No.  2)  develop  they  may  still  be  so  numerous  as  to 
give  rise  to  a  mass  culture.  Should  only  a  few  bacteria  be 
present  they  will  be  scattered  over  the  surface,  separated 
by  an  appreciable  distance,  and,  when  they  develop,  they 
will  give  rise  to  isolated  growths  or  colonies.  Inasmuch 
as  the  result  with  the  second  potato  is  uncertain  it  is  cus- 
tomary to  take,  by  means  of  a  sterile  knife,  a  small  portion 
of  material  from  the  surface  of  potato  No.  2,  and,  to  spread 
this  over  that  of  a  third  potato  (No.  3).  The  latter  will 
probably  have  planted  on  its  surface  some  10  or  20  bacteria. 
These  may  be  i  to  |  an  inch  apart.  The  bacterial  cell, 
wherever  deposited,  grows  and  multiplies,  and  eventually 
forms  a  visible,  pin-head  growth.  Ttyis  isolated  growth  is 
known  as  a  colony,  and,  inasmuch  as  it  is  derived  from  a 
single  cell,  it  is  a  pure  culture  of  that  organism. 

By  resorting  to  dilution  cultures,  in  the  manner  indi- 
cated, it  is  possible  to  isolate  the  various  bacteria  that  may 
be  present  in  the  original  material.  To  illustrate  the  dilu- 
tion that  takes  place  it  may  be  assumed  that  10,000,000  cells 
were  planted  on  potato  No.  1.  The  small  portion  taken 
from  this  may  have  planted  10,000  bacteria  on  potato  No.  2. 
Similarly,  the  small  amount  taken  from  the  second  and 
planted  on  the  surface  of  the  third  potato  may  not  contain 
more  than  10  cells.  In  36  to  48  hours,  if  the  temperature  is 
favorable,  these  cells  will  have  multiplied  to  such  an  extent 
as  to  give  rise  to  visible  growths  or  colonies. 

The  simple  method  of  dilution,  as  just  described,  is  the 


170  BACTERIOLOGY. 

basis  of  all  bacteriological  work.  The  potato  was  the  first 
medium  employed  for  isolating-  bacteria.  In  time,  other 
media  were  introduced,  but  the  fundamental  principle,  that 
of  physical  dilution,  is  the  same,  regardless  of  the  medium 
employed. 

In  this,  and  all  subsequent  work,  successful  results  and 
freedom  from  personal  danger  depend  upon  the  rigid  steril- 
ization of  all  articles  used.  Attention  to  the  smallest  de- 
tail is  necessary  in  order  to  prevent  contaminations.  The 
training,  thus  acquired,  will  be  invaluable,  in  time,  in  the 
intelligent  prevention  of  disease.  The  following  rule  can- 
not, therefore,  be  emphasized  too  strongly:  Sterilize  all  in- 
struments, wires,  etc.,  immediately  before  use  and  immediately 
after  use.  Instruments  are  sterilized  immediately  before  use 
in  order  to  avoid  contaminations;  and  immediately  after  use, 
in  order  to  prevent  personal  danger.  This  rule  should  be 
rigidly  adhered  to.  Careless  manipulations,  acquired  while 
engaged  in  the  study  of  non-pathogenic  bacteria,  may  be 
unconsciously  resorted  to  when  studying  disease-producing 
organisms.  A  careless  student  is  a  source  of  danger,  not  only 
to  himself  but  also  to  his  neighbors.  The  sterilization  of 
all  instruments  should  be  attended  to,  before  they  are  placed 
back  on  the  table. 

Certain  additional  personal  precautions  should  be  ob- 
served, while  engaged  at  work  in  the  laboratory.  Pencils, 
pens,  glass  rods  and  labels  should  never  be  introduced  into 
the  mouth.  The  tumblers,  or  glasses  in  the  desk  should  not 
be  used  for  drinking  purposes.  If  any  material  drops  on  the 
table  or  floor,  it  should  at  once  be  rendered  harmless  by 
covering  it  with  the  mercuric  chloride  solution.  Invariably, 
at  the  close  of  the  day's  work,  the  table  should  be  washed 
with  this  solution  and,  at  the  same  time,  the  hands  should 
be  disinfected. 

Laboratory  work.—  Make  a  dilution  culture,  as  described  (on  three 
potatoes),  of  the  Bacillus  prodigiosus. 


GELATIN  AND  POTATO  MEDIA.  171 

Make  a  mass  culture  (single  potato)  of  each  of  the  following: 
Orange  sarcine,  Red  bacillus  of  water,  Violet  bacillus  of  water. 

The  inoculated  potatoes  are  kept  in  the  moist  chamber,  which 
should  be  set  aside  where  the  sun  will  not  strike.  When  moisture  ac- 
cumulates on  the  under  side  of  the  lid,  it  should  be  removed  at  once, 
in  the  manner  indicated.  On  the  second  day,  the  cultures  are  exam- 
ined in  hanglng--drops,  and  on  the  day  following-  they  are  used  for 
staining-  purposes. 

For  modifications  of  the  above  method  of  making-  cultures  on  po- 
tato see  p.  182. 

Gelatin  Plate  Culture. 


It  has  been  mentioned  that  cultivation  on  potatoes  was 
resorted  to  at  a  very  early  date.  The  far-reaching1  studies 
of  Pasteur  led  to  the  introduction  of  various  liquid  media, 
such  as  beef,  veal,  or  chicken  broth.  Nutrient  solutions, 
containing-  various  mineral  salts,  were  likewise  employed. 
In  order  to  isolate  bacteria,  which  might  be  present  in  a 
mixture,  it  was  necessary  to  resort  to  excessive  dilution, 
and,  even  then,  it  was  far  from  certain  that  a  pure  culture 
was  really  obtained.  One  of  the  greatest  aids  to  the  ad- 
vancement of  bacteriology  was  supplied  by  Koch,  when  he 
introduced  the  use  of  gelatin.  By  the  addition  of  gelatin 
to  beef  tea,  a  transparent  medium  was  obtained  which  was 
solid  at  ordinary  temperature,  and  which  could  be  liquefied 
without  any  difficulty.  This  transformation  of  a  liquid  me- 
dium into  a  solid  one  rendered  it  possible  to  isolate  bacteria 
in  a  condition  of  absolute  purity.  The  uncertainty  regard- 
ing the  action  of  bacteria,  whether  they  themselves  pro- 
duced certain  changes,  or  whether  it  was  due  to  something 
else  that  accompanied  them,  was  thus  easily  set  aside. 

The  object  of  the  gelatin  plate  method,  as  with  the 
dilution  potato  culture  already  made,  is  to  isolate  the 
several  kinds  of  bacteria  that  may  be  present  in  a  mixture. 
The  isolated  organisms  developing  in  a  solid,  transparent 
medium,  form  colonies  which  are  easily  perceived,  and 


172  BACTERIOLOGY. 

from  which  transplantations  can  be  readily  made.  Colon- 
ies of  bacteria  which  would  escape  detection  when  growing1 
on  the  surface  of  a  potato,  because  they  are  invisible,  are 
easily  seen  in  the  solid,  transparent  layer  of  g'elatin.  Pure 
cultures,  of  the  different  kinds  of  bacteria,  are  thus  ob- 
tained. 

Method. — First,  sterilize  six  glass  plates  (13  X  10  cm.) 
by  placing1  them  in  an  iron  box,  and  heating1  this  in  the  dry- 
heat  sterilizer,  at  a  temperature  of  150-175°,  for  >^-^ 
of  an  hour.  A  temperature  of  170°  maintained  for  15  min- 
utes, will  be  sufficient  (see  p.  160).  Remove  the  box,  and  let 
it  cool  in  the  room  at  the  ordinary  temperature. 


FIG.  28.  Apparatus  used  in  cultivation,  a — Glass  benches  and  plates;  b — Moist 
chamber  for  plates;  c — Petri  dish;  d — Esmarch  dish;  e — Platinum  wires,  straight  and 
with  loop,  fused  into  the  ends  of  glass  rods. 

In  the  meantime,  prepare  the  necessary  platinum  wires.  These 
should  be  about  5  cm.  (2  in.)  in  length,  and  in  thickness  should  corre- 
spond to  about  gauge  number  22  (0.5  mm.).  The  end  of  a  glass  rod, 
about  18  cm  (7  in.)  long  and  6  mm.  (%  in.)  wide,  is  heated  in  a  Bunsen 
flame,  or  better  still,  in  a  blast  lump,  till  it  has  become  quite  soft. 
The  platinum  wire  is  then  pushed  into  the  soft  end.  If  a  depression 
occurs  around  the  wire,  where  it  enters  the  glass,  this  can  be  easily 
remedied  by  gently  pulling  on  the  wire,  while  the  glass  is  still  soft. 
A  glass  tube  should  never  be  used  for  mounting  platinum  wires. 

In  order  to  prevent  sudden,  uneven  cooling,  it  is  well  to  partially 
anneal  the  glass,  by  allowing  a  small  luminous  flame  to  deposit  a 
layer  of  lamp-black  on  the  hot  end.  This  is  then  wiped  off  when  the 
glass  is  perfectly  cool.  It  is  well  to  have  on  hand  three  mounted, 
platinum  wires.  The  wire  should  be  kept  perfectly  straight,  not 
crooked.  When  plating  or  doing  similar  work,  the  end  is  bent  into  a 
small  loop  which  has  a  clear  diameter  of  about  2  mm.  (Fig.  28  e). 


GELATIN   AND  POTATO   MEDIA.  173 

The  platinum  wires  are  sterilized  by  holding-  them  in 
the  flame,  till  incandescent.  The  end  of  the  glass  rod 
should  always  be  thoroughly  heated,  and,  if  any  organic 
matter  is  present,  such  as  gelatin,  it  should  be  burned  off. 
The  wires  should  be  kept  in  a  conical  test-glass  (Fig.  43), 
in  the  bottom  of  which  is  some  cotton. 


FIG.  29.    The  inoculation  of  a  single  tube. 

Place  three  of  the  sterilized,  gelatin  tubes  in  a  water- 
bath  which  has  been  warmed  to  about  30-35°.  When  the 
gelatin  melts,  the  tubes  are  ready  for  inoculation.  With  a 
sterilized,  cooled  platinum  wire,  pick  up  a  minute  amount, 
of  the  growth  on  potato,  of  the  Bacillus  prodigiosus.  Place 
one  of  the  liquefied  g'elatin  tubes  between  the  thumb  and 
index  finger  of  the  left  hand,  so  that  it  is  almost  horizontal. 
It  is  held  in  this  position  in  order  to  prevent  foreign  matter 
from  dropping  into  the  tube.  The  neck  of  the  tube  with  its 
plug,  as  well  as  the  palm  of  the  left  hand,  is  turned  to  the 
right.  In  this  position  the  entire  length  of  the  tube  is  be- 
fore one's  eyes  (Fig.  29).  While  still  holding  the  platinum 
wire  in  the  right  hand,  grasp  the  cotton  plug  with  the  little 
finger  of  that  hand,  and,  remove  it  by  slight  rotation.  If 
any  tufts  of  cotton  are  adherent  to  the  neck  of  the  tube, 
touch  it  for  a  moment  to  a  Bunsen  flame.  Otherwise,  the 
platinum  wire  might  carry  in  some  of  this  material,  and 
thus  cause  unnecessary  contamination.  As  a  matter  of  rou- 
tine, it  is  well  to  always  flame  the  'neck  of  a  tube  before  insert- 


174 


BACTERIOLOGY. 


ing  the  wire.  Now  pass  the  inoculated  wire  into  the  tube, 
and  thoroughly  mix  the  bacteria  thus  introduced,  with  the 
gelatin.  Then  withdraw  the  wire,  replace  the  cotton  plug", 
and  sterilize  the  platinum  wire  in  a  flame. 

With  a  colored  wax  pencil,  mark  the  tube  thus  inocu- 
lated, 1.  Likewise,  mark  another  liquefied  gelatin  tube,  2. 
Place  tube  1  in  the  left  hand,  in  the  same  position  as  be- 
fore, and  then  place  next  to  it  tube  2  (Fig.  30).  Remove 
the  cotton  plug  of  tube  2,  and  place  it  between  the  adjoin- 
ing index  and  middle  fingers.  Then  remove  the  cotton  plug 
of  tube  1,  -and  place  it  between  the  ring  and  little  finger. 
Touch  the  flame  of  a  burner  to  the  necks  of  the  tubes,  in 
order  to  burn  off  any  loose  cotton.  Now,  with  a  sterilized, 
cooled  platinum  wire,  the  end  of  which  is  provided  with  a 
small  loop,  transfer  a  loopf ul  of  gelatin  from  tube  1  to  tube 
2,  and  mix  well.  Return  the  same  platinum  wire  to  tube  1, 
and  again  transfer  a  loopful  of  gelatin  to  tube  2.  Repeat 
this  once  more,  so  that  all  told,  three  transfers  of  inocu- 
lated gelatin  have  been  made.  Replace  the  cotton  plugs 
in  their  respective  tubes,  sterilize  the  platinum  wire  and  set 
the  tubes  in  a  tumbler,  the  bottom  of  which  is  covered  with 
a  layer  of  cotton. 


FIG.  30.    Inoculation  from  tube  to  tube  when  diluting. 


Mark  another  liquefied  gelatin  tube,  3.  Then  place  2 
in  the  position  in  which  No.  1  was  just  held,  and  next  to  it 
place  tube  3.  Remove  the  cotton  plugs,  place  in  their  re- 
spective positions,  and  flame  the  necks  of  the  tubes  as 


GELATIN   AND   POTATO  MEDIA.  175 

before.  With  a  sterilized,  cooled  platinum  wire,  make  three 
successive  transfers  of  gelatin  from  tube  2  to  tube  3.  Re- 
turn the  cotton  plug's  to  their  tubes,  sterilize  the  wire,  and 
set  the  tubes  aside  in  the  tumbler. 

Each  of  the  three  gelatin  tubes  has  now  been  inocu- 
lated. Tube  1  usually  has  a  very  large  number  of  bacteria, 
while  tube  2  has  less  and  tube  3  should  have  but  a  small 
number,  so  that,  subsequently,  when  colonies  develop 
these  should  be  separated  from  one  another  by  an  appre- 
ciable distance.  This  successive  dilution,  it  will  be  ob- 
served, corresponds  to  that  performed  when  making  potato 
cultures.  It  is  necessary  to  take  a  very  minute  amount  of 
material  for  the  inoculation  of  tube  1  in  order  to  to  obtain 
good  dilutions.  Moreover,  working  as  the  class  does  with 
pure  cultures,  it  will  be  sufficient  to  make  transfers  of  only 
one  or  two  loopsful  of  gelatin,  instead  of  three. 

In  transferring  gelatin  from  one  tube  to  another,  care 
must  be  taken  to  prevent  the  wire  from  coming  into  contact 
with  the  neck,  or  wall  of  the  tube.  Unnecessary  contact 
of  the  end  of  the  glass  rod  with  gelatin  should  be  avoided, 
and,  if  gelatin  does  cover  the  end  it  should  be  burned  off 
completely. 

The  ice-plating-  apparatus  should  now  be  prepared  for  use. 
Broken  ice  is  placed  in  the  glass  dish,  which  is  then  filled  with  water, 
so  that  when  the  plate  is  placed  in  position  it  rests  directly  on  the 
ice  water.  No  air-space  should  be  present.  The  apparatus  should  be 
levelled  and  is  then  ready  for  use  (Fig-.  31).  Instead  of  the  ordinary 
plating-  apparatus,  one  similar  to  that  shown  in  Fig".  32  can  be  em- 
ployed. It  is  made  of  g-alvanized  iron  or  of  copper,  and  is  15  cm. 
(6  in.)  hig-h.  The  inflow  tube  (1  cm.)  extends  along-  the  bottom  to  the 
farther  end  of  the  box.  The  wide  outflow  tube  (2  cm.)  does  not  extend 
inward.  Tap-water  is  allowed  to  flow  into  the  apparatus.  It  runs  the 
entire  length  of  the  box  and  thus  reduces  the  temperature  of  the 
surface  plate  to  that  of  the  water  (about  15°).  The  box  is  68.5  cm. 
(27  in.)  long-,  28  cm.  (11  in.)  wide,  and  7.5  cm.  (3  in.)  deep. 

Remove  a  sterilized  glass  plate  from  the  iron  box,  by 
grasping  the  edges  with  two  fingers;  place  it  upon  the 


176 


BACTERIOLOGY. 


ground  plate  of  the  ice  apparatus,  and  cover  with  the  bell- 
jar.  As  soon  as  the  plate  is  cool,  it  is  ready  to  receive  the 
gelatin.  Before  pouring1  the  contents  of  a  tube  upon  a 
plate,  it  is  necessary,  as  a  matter  of  precaution,  to  sterilize 
the  neck  of  the  tube.  This  is  easily  accomplished,  in  the 
following"  manner:  Remove  the  cotton  plug"  from  the  tube 
(No.  1),  and  rapidly  rotate  the  neck  of  the  tube  in  the 
flame.  This  is  thus  sterilized,  and  the  contents  of  the  tube 
can  now  be  poured,  without  coming  into  contact  with  for- 
eign organisms.  In  about  a  minute,  the  neck  of  the  tube 
is  sufficiently  cool  to  proceed.  Raise  the  bell-jar  some- 
what, shielding  the  plate  as  much  as  possible  from  draughts 
of  air,  and  pour  the  gelatin  on  the  center  of  the  plate. 
With  the  lip  of  the  tube,  rapidly  spread  the  gelatin  over 
as  much  of  the  surface  as  possible,  avoiding,  however,  the 
edges  of  the  plate.  The  plate  is  now  allowed  to  remain 

under  the  bell-jar 
till  the  gelatin  be- 
comes solid.  The 
empty  gelatin  tube 
should  not  be  placed 
on  the  table  but 
should  be  set  in  a 
tumbler.  When 
through  plating,  all 
the  empty  tubes 
should  be  sterilized, 
either  by  immersion 
in  mercuric  chlo- 
ride, or  better  by 
exposure  to  boiling  water  or  to  steam. 

While  the  gelatin  on  the  plate  is  becoming  solid,  a 
"moist  chamber  "  is  prepared  in  the  same  way  as  for  potato 
cultures  (p.  167).  It  is  not  necessary  to  sterilize,  either  the 
moist  chamber,  or  the  glass  benches  on  which  the  plates 
are  to  be  placed.  Instead  of  mercuric  chloride,  tap-water 


FIG.  31.    Ice  apparatus  for  cooling  gelatin  plates. 


I 


GELATIN   AND  POTATO  MEDIA. 


177 


may  be  used  to  moisten  the  paper  which  covers  the  bottom 
of  the  dish.  On  three  small  pieces  of  paper,  write 
the  name  of  the  germ  or  material,  the  number  of 
the  plate,  and  the  date.  This  label  may  be  written 
on  the  giass  bench  (Fig-.  28  a).  Now,  place  a  glass 
bench  on  the  bottom  of  the  moist  chamber,  and,  on 
it  the  label  for  plate  1.  Transfer  the  gelatin  plate  from 
the  ice  apparatus  to  the  bench.  Pour  the  contents  of  the 
remaining1  gelatin  tubes  on  plates,  in  the  same  manner  as 
described;  and,  when  cool,  transfer  to  the  benches  which 
are  arranged  one  above  the  other,  in  the  moist  chamber.  In 
doing  this,  the  utmost  care  should  be  taken  to  avoid 
touching  the  gelatin,  either  with  the  fingers  or  with  a 
glass  bench.  Each  chamber  can,  or  should  hold  a  stack 
of  six  plates. 


FIG.  32.    Water  apparatus  for  cooling  plates  (F.  G.  N.). 

The  moist  chamber  is  now  set  aside.  It  should  not  be 
placed  near  the  steam  coil,  or  where  the  sun-light  may  pos- 
sibly strike  it.  The  rapidity  with  which  colonies  develop 
in  the  gelatin  will  depend  upon  the  temperature  of  the  room, 
and,  to  a  certain  extent,  upon  the  organism  itself.  The  best 
temperature  for  the  development  of  a  growth  on  gelatin 
plates  is  about  18-20°.  Under  this  condition,  excellent  col- 
onies will  form,  as  a  rule,  in  from  36  to  48  hours.  The  lower 
the  temperature  the  slower  will  be  the  development.  If  the 
temperature  exceeds  20°  the  growth  will  be  more  rapid,  but, 
in  that  case,  there  is  always  danger  of  melting  the  gelatin 
and  thus  spoiling  the  outcome.  The  ordinary,  10  per  cent. 

12 


178  BACTERIOLOGY. 

gelatin  melts  at  about  25°.  In  warm  weather,  as  in  sum- 
mer, gelatin  plates  cannot  be  developed  at  the  prevailing 
room  temperature.  In  order  to  keep  the  gelatin  solid,  it  is 
customary  in  that  case  to  set  the  plates  in  an  ice-chest. 
Inasmuch  as  the  temperature  in  the  ice-chest  is  about  10°, 
the  growth  of  the  colonies  will  be  materially  retarded,  and 
this  at  times  may  lead  to  annoying  delays. 

The  nutrient  gelatin,  as  commonly  prepared,  contains 
10  per  cent,  of  gelatin.  Unless  otherwise  specified,  this  is 
assumed  to  be  the  composition  of  the  medium  when  "gela- 
tin "  is  mentioned.  Occasionally  a  nutrient  medium  con- 
taining 15  per  cent,  of  gelatin  is  employed.  This  melts  at  a 
higher  temperature  than  does  the  ordinary  gelatin,  and 
hence,  is  specially  made  use  of  during  warm  weather.  It 
should  be  borne  in  mind,  however,  that  the  cultural  charac- 
teristics of  an  organism  may  vary  considerably  with  the 
hardness  of  the  medium  in  which  it  is  grown.  Atypical  cul- 
tures will  not  infrequently  be  obtained  when  an  organism  is 
grown  on  hard  gelatin. 

The  apparatus  shown  in  Fig.  33  is  employed  in  this  laboratory, 
to  cultivate  bacteria  at  a  constant  low  temperature,  regardless  of  that 
which  prevails  in  the  room.  It  will  be  found  as  useful  for  this 
purpose,  as  the  incubator  is  for  higher  temperatures.  It  has  the 
merit  of  being  simple  and  inexpensive.  The  apparatus  is  made  of 
galvanized  iron  or  of  copper.  The  outer  box  is  22  cm.  high,  68  cm. 
long  and  35  cm.  wide,  while  the  inner  compartment  is  18  x  53  x  27  cm. 
The  narrow  tube  (1.2  cm.  diameter)  to  the  left  stops  short,  on  the 
inside  of  the  box.  It  is  connected  directly  with  the  water-supply  pipe, 
and  not  by  means  of  a  narrow  faucet.  Next  to  this  inflow  is  the  wide 
outflow  tube  (2  cm.  diameter),  which  passes  along  the  bottom  of  the 
box  to  the  opposite  end,  where  it  is  turned  upward.  A  short  rubber 
tube  is  slipped  over  the  end  of  the  upright  tube,  and,  by  moving  it  up 
or  down,  the  height  of  the  water  can  be  regulated  at  will.  The  bent 
wide  tube  on  the  side  is  a  safety  over-flow.  The  inner  box  is  held  in 
place  and  kept  from  floating,  by  means  of  two  stout  rods.  The  water 
flows  into  the  large  box,  under  and  around  the  inner  compartment, 
and  then  leaves  by  the  outflow  and  overflow  tubes. 

The  temperature  in  the  inner  compartment  will  depend  upon  the 
rate  of  flow  of  the  water,  and  on  the  temperature  of  the  water.  With 


I 


GELATIN  AND  POTATO  MEDIA.  179 

a  maximum  flow  the  temperature  in  the  inner  compartment  will  be 
the  minimum  attainable.  In  this  laboratory  the  temperature  of  the 
water  during-  summer,  as  it  comes  from  the  ground,  is  14-15°.  This 
represents,  then  the  lowest  constant  temperature  attainable.  By 
diminishing-  the  flow  of  the  water,  heat  is  absorbed,  and  a  hig-her 
temperature  can  be  obtained.  The  inner  compartment  can  thus  be 
maintained  at  18°,  20°  or  22°.  In  the  apparatus,  as  actually  used,  the 
flow  is  such  that  a  temperature  of  18-20°  is  maintained  throughout 
the  summer  months.  It  is  well  to  keep  inside  of  the  apparatus  a 
Kappeler  maximum  and  minimum  thermometer. 


FIG.  33.    The  author's  constant,  low  temperature  apparatus. 

Laboratory  work.— The  student  will  make  plate  cultures  of  the 
Bacillus  prodigiosus  and  of  Bacillus  Indicus.  The  potato  cultures 
made  heretofore  will  supply  the  material  for  inoculation. 

Modified  Gelatin  Plate  Cultures. 

The  original  plate  method  of  Koch,  as  described  on 
p.  17*2,  has  been  subjected  to  several  modifications,  some  of 
which  may  be  considered  as  decided  improvements.  The 
iron  box,  plates,  benches,  moist-chambers  and  levelling-  ap- 


180  BACTERIOLOGY. 

paratus,  are  more  or  less  expensive,  and  are  too  cumber- 
some to  be  transported  from  place  to  place,  in  the  case  of 
out-door  investigations.  Even  in  the  laboratory,  the  latter 
apparatus  requires  time  to  prepare  and  to  level  it,  and,  not 
infrequently,  ice  may  be  lacking1.  Furthermore,  the  moist- 
chamber  when  placed  in  an  incubator,  or  in  a  cooler,  takes 
up  an  unnecessary  large  amount  of  space.  In  the  plate 
method  as  given,  more  or  less  contamination  may  result 
from  exposure  to  the  air,  while  the  plates  are  on  the  ice 
apparatus,  or  subsequently,  when  they  are  kept  in  the 
large  moist  chamber.  Whenever  it  is  desired  to  examine 
the  growth  on  the  plates,  it  is  necessary  to  expose  these 
freely  to  the  air.  Even  if  but  one  plate  is  to  be  examined, 
it  necessitates  the  exposure  of  all  to  contamination  from 
the  air.  Furthermore,  it  may  happen  that  the  gelatin  on 
an  upper  plate  liquefies,  either  by  heat  or  by  the  growing 
bacteria,  and  then  drips  over  the  edge  of  the  plate  on  those 
which  are  below  it.  To  overcome  these  difficulties,  Petri 
introduced  the  use  of  the  shallow  dishes  (Fig.  28  c)  which 
bear  his  name.  The  cover  to  the  dish  should  be  10  cm.  in 
diameter,  and  the  bottom  dish  should  be  as  flat  as  possible, 
and  should  not  be  more  than  1  cm.  deep.  A  greater  depth  in- 
terferes in  the  subsequent  examination  with  the  microscope. 

Petri  dish  culture. — The  Petri  dishes  (Fig.  28  c)  are  placed 
in  a  wire  basket,  and  sterilized  in  the  dry-heat  oven  (Fig. 
25),  and  then  allowed  to  cool.  If  the  dishes  are  to  be  kept 
for  some  time  before  use,  or  are  to  be  transported,  they 
.should  be  wrapped  in  paper,  and  then  sterilized.  Three 
gelatin  tubes  are  inoculated  with  the  organism  to  be 
plated,  the  dilutions  being  made  in  exactly  the  same  man- 
ner as  described  in  connection  with  the  ordinary  plate 
method  (p.  174).  The  contents  of  each  tube  are  then 
poured  into  one  of  the  cooled,  sterile  Petri  dishes.  The 
precaution  of  flaming  the  neck  of  the  tube  (p.  176)  before 
pouring  out  the  gelatin  must,  of  course,  be  observed. 


I 


GELATIN  AND  POTATO  MEDIA.  181 

When  pouring-  the  gelatin,  the  cover  of  the  Petri  dish 
^hould  not  be  raised  any  more  than  is  absolutely  necessary. 
The  cover  is  then  replaced,  and  the  dish  is  gently  tilted, 
from  side  to  side,  so  as  to  cause  the  gelatin  to  spread  over 
the  entire  bottom.  The  dishes  are  then  placed  on  a  flat 
surface,  and,  when  the  gelatin  has  solidified,  they  are  set 
aside  in  order  to  allow  the  colonies  to  develop. 

In  order  to  make  a  preliminary  examination  of  the 
colonies,  the  dish  is  inverted  on  the  stage  of  the  micro- 
scope. The  colonies  can  thus  be  studied  almost  as  well  as 
if  the  top  was  removed.  In  order  to  make  transplantations 
from  the  colonies,  it  will  be  necessary,  of  course,  to  remove 
the  top,  and  to  place  the  bottom  upright  on  the  stage  of 
the  microscope. 

In  this  method,  each  dish  constitutes  a  plate  and  a 
moist  chamber.  The  progress  in  development  can  be  ob- 
served without  exposing  the  gelatin  to  the  air,  and  with- 
out any  risk  to  the  other  plates.  Consequently,  the  chance 
of  contamination  is  reduced  to  a  minimum.  There  is  little, 
or  no  danger  of  the  gelatin  dripping  on  the  floor,  table,  or 
stage  of  the  microscope.  Moreover,  the  use  of  the  ice  ap- 
paratus, of  plates,  benches,  boxes,  etc.,  is  done  away  with. 
This  method  of  obtaining  colonies  is  to  be  used,  whenever 
possible,  in  preference  to  the  original  Koch  method. 

Esmarch  roll-tube  method. — In  this  the  advantages  of  the 
plate  method  are  secured  without  the  use  of  any  extra  ap- 
paratus, such  as  plates  or  dishes.  The  inoculated  gelatin, 
instead  of  being  poured  out  on  sterilized  plates,  or  into 
dishes,  is  solidified  in  a  thin  film  on  the  inside  wall  of  the 
test-tube.  Another  advantage  of  this  method  is,  that  it  is 
well  adapted  for  those  organisms  which  grow  very  slowly, 
and  require  a  week  or  two  to  form  distinct  colonies.  Desic- 
cation of  the  gelatin  can  be  readily  prevented  in  the  roll- 
tube,  whereas  this  is  more  difficult  to  do  in  plate,  or  in 
dish  cultures. 


182  BACTERIOLOGY. 

Three  gelatin  tubes  are  inoculated  in  the  usual  manner 
(p.  174)  with  the  material  to  be  plated.  It  is  advisable  to 
select  very  wide  tubes.  The  cotton  projecting-  from  the 
tube  is  then  cut  off,  and  the  neck  of  the  tube  is  rotated 
rapidly  in  a  flame  in  order  to  insure  sterilization  of  the 
outer  layer  of  cotton,  since  this  may  subsequently,  come 
into  contact  with  the  gelatin.  The  cotton  plug  is  then 
pushed  in  a  trifle,  and  the  tube  is  closed  by  means  of  a  rub- 
ber cap.  A  boiled  cork  or  rubber  stopper  may  be  used  for 
this  purpose.  It  is  then  immersed  in  cold,  or  better,  in  ice- 
water,  and  is  rotated  slowly  in  an  almost  horizontal  posi- 
tion. The  gelatin  should  solidify  in  a  thin,  even  layer, 
over  the  inner  wall  of  the  tube.  During  the  process  of  roll- 
ing, care  should  be  taken  to  prevent  the  gelatin  from  com- 
ing into  contact  with  the  cotton  plug.  It  will  not  only 
cause  the  cotton  to  adhere  to  the  glass,  but  may  prevent 
access  of  air,  and  thus  bring  about  partial  anaerobic  condi- 
tions within  the  tube. 

A  better  method  of  rolling  the  tube  is  to  do  this  on  a 
block  of  ice.  A  very  slightly  inclined  groove  can  be  made 
in  the  ice  by  means  of  a  test-tube  filled  with  hot  water.  In 
this  case  it  is  unnecessary  to  heat  the  cotton,  or  to  use  a 
rubber  cap  or  a  stopper.  The  roll-tubes  should  be  set  aside 
in  a  cool  place  (15°),  to  prevent  collapse  of  the  gelatin. 
The  colonies  are  then  examined  under  the  microscope  and 
transplantations  can  be  readily  made  from  these,  especially 
if  the  tube  is  wide.  For  transplanting,  a  platinum  wire 
should  be  used,  the  end  of  which  is  bent  at  right  angles. 

Laboratory  work.— The  student  will  make  Esmarch  roll  tubes  of  Yel- 
low sarcine,  and  Petri  dish  cultivations  of  the  Orange  sarcine. 

Modified    Potato  Cultures. 


The  method  of  making  potato  cultures,  as  described  on 
p.  167,  is  open  to  many  of  the  same  objections  which  were 
made  in  connection  with  the  ordinary  gelatin  plate  method. 


GELATIN  AND   POTATO  MEDIA.  183 

It  has,  consequently,  given  place  to  two  modifications  which 
give  excellent  results.  One  or  the  other  of  these  should, 
therefore,  be  employed  whenever  it  is  desired  to  obtain 
strictly  pure  cultures  on  potatoes. 

Esmarch  potato  cultures. — The  cover  of  the  Esmarchdish 
(Fig.  28  d),  is  about  5  cm.  in  diameter,  and  the  bottom  is  2 
cm.  in  height.  These  dishes,  like  those  of  Petri,  are  steril- 
ized in  the  dry-heat  oven  (Fig.  25,  p.  160),  and  then  allowed 
to  cool. 

A  small,  sound  potato  is  selected,  and  held  with  the 
thumb  and  forefinger  of  the  left  hand.  The  outer  edg^e  of 
the  potato  is  pared  circularly,  by  means  of  a  clean  potato- 
knife  which  is  held  upright.  Two  horizontal  sections,  above 
and  below,  are  now  made.  The  clean,  sound  core  of  the  po- 
tato, which  is  thus  obtained,  is  slipped  into  a  sterilized  Es- 
march dish.  Each  dish  is  thus  supplied  with  a  clean  potato 
section.  The  dishes  are  then  placed  in  a  steam  sterilizer 
and  steamed  for  £  to  1  hour.  The  potatoes  will  then  be 
sterilized  and  cooked.  It  is  not  necessary  to  heat  the  pota- 
toes on  three  successive  days.  The  potatoes  will  become 
more  or  less  dark,  unless  they  are  heated  immediately  after 
they  are  cut. 

Laboratory  work. — The  student  will  prepare  three  of  the  Esmarch 
potato  dishes,  and  then  make  dilution  cultures  of  the  Bacillus  prodi- 
giosus.  As  little  of  the  material  as  possible  should  be  transferred, 
each  time,  and  it  should  be  spread  over  the  entire  surface.  The 
method  of  inoculation  is  the  same  as  that  described  on  p.  168,  except 
that  the  potatoes  are  not  taken  out  of  the  dishes.  When  dilution 
cultures  are  not  desired,  a  single  mass  culture  may  be  made,  or  sev- 
eral parallel  streaks  may  be  made  by  means  of  a  platinum  wire.  In 
the  latter  case,  the  organism  will  develop  along-  the  streaks  thus 
made.  Inasmuch  as  each  potato  is  in  a  small  sterilized  dish  by  itself, 
the  risk  of  contamination,  with  careful  and  rapid  manipulation,  is 
very  small. 

Test-tube  cultures  on  potato. — In  this  method,  the  special 
dish  of  Esmarch  is  done  away  with  and  its  place  is  taken 


184 


BACTERIOLOGY. 


by  a  test-tube.  The  method  was  introduced  about  the  same 
time  by  Bolton  in  this  country,  Globig  in  Germany,  and 
Roux  in  France.  It  is  the  method  which  is  most  often 
employed  when  studying-  the  cultural  characteristics  on 

potato,  of  an  organism.  This  is 
because  it  is  convenient  and 
simple  in  execution,  and,  like  all 
tube  cultivations,  almost  entirely 
free  from  danger  of  contamina- 
tion. 

The  large  test-tubes  (15  x  150 
mm. )  should  be  employed.  These 
are  cleaned,  plugged,  and  steri- 
lized in  the  usual  way.  A  cork- 
borer  should  then  be  selected 
which  will  easily  pass  into  these 
tubes.  In  the  absence  of  a  cork- 
borer,  the  student  can  easily  im- 
provise one  out  of  a  narrow  test- 
tube.  For  this  purpose  a  sharp 
file-mark  is  made,  about  three- 
fourths  of  an  inch  from  the  end. 
The  end  of  a  glass  rod,  which 
has  been  heated  in  the  flame  of 
a  burner,  or  blast-lamp,  till  red- 
hot,  is  then  touched  to  the  edge 
of  the  file-mark.  The  glass 
cracks,  and  leaves  a  clean  cut 
end.  A  piece  of  ignited  char- 

FIG.  34.     Tubes  for  cultivation  on 

potato.    ^-Ordinary  form;    b— ROUX  coal  is  better  than  the  glass-rod 

form. 

since,  by  gently  blowing,  it  can 
be  kept  red-hot,  while  being  held  against  the  glass. 

Several  large  potatoes  are  brushed,  under  the  tap,  and 
then  steamed,  or  immersed  in  boiling  water  for  20  minutes. 
A  number  of  cylinders  are  then  punched  out  of  the  cooled 
potatoes.  These  cylinders  (4-5  cm.  long)  should  be  placed 


GELATIN  AND  POTATO  MEDIA.  185 

on  a  clean  piece  of  paper;  the  ends  should  then  be  trimmed 
off  at  rig-ht  angles,  and  finally  the  cylinders  should  be  cut 
diagonally  in  two.  The  potato  wedges,  thus  obtained,  are 
now  slipped,  with  the  wide  end  downward,  into  sterilized 
tubes  (Fig.  34  a).  The  cut  surface  of  the  potato  is  inclined, 
and  hence  can  be  readily  inoculated  by  means  of  a  platinum 
wire.  The  potato  tubes,  thus  prepared,  can  be  submitted 
to  fractional  sterilization  in  steam,  being  heated  for  about 
20  minutes  each  day  on  three  successive  days.  Or,  they 
may  be  given  a  single  steam  sterilization  of  ^-1  hour. 

The  method,  as  given  above,  will  yield  potato  cylin- 
ders which  are  pure  white,  and  will  remain  so.  If  a  raw 
potato  is  punched  or  cut,  the  cut -surf  ace,  as  a  result  of  ox- 
idation, will  soon  take  on  a  light  pink  tinge  and,  on  subse- 
quent heating,  this  turns  to  a  dirty  gray.  This  alteration 
in  the  appearance  can  be  avoided  by  at  once  submitting  the 
cut  potato  to  the  action  of  steam.  If  the  potato  has  been 
cooked  before  hand,  it  will  not  oxidize  pn  subsequent  ex- 
posure. 

Instead  of  the  ordinary  test-tube,  Roux  employed  one 
which  was  provided  with  a  slight  constriction  near  the  end 
(Fig.  34  b).  The  wedge  of  potato,  in  this  case,  does  not  rest 
on  the  bottom  of  the  tube,  but  on  the  constricted  portion- 
The  space  below  this  may  be  filled  with  water,  or  with  any 
desired  liquid.  The  surface  of  the  potato  can,  therefore, 
be  moistened,  whenever  it  is  desirable  to  do  so.  When  a 
five  per  cent,  solution  of  glycerin  is  employed,  the  potato 
becomes  an  excellent  soil  for  the  growth  of  the  tubercle 
bacillus. 

The  Roux  potato  tube  is  consequently  very  useful.  It  can  be 
prepared  by  the  student  without  the  least  difficulty.  The  clean, 
sterile,  wide  test-tubes  should  be  used.  A  narrow  flame  from  the 
blast-lamp  should  be  directed  horizontally,  against  the  test-tube,  at 
about  one  inch  from  the  bottom.  The  tube  should  be  held  vertically, 
and  rotated  slowly,  so  that  the  point  of  the  flame  just  strikes  it. 
The  glass  gradually  softens  and  the  constriction  results. 


186  BACTERIOLOGY. 

The  inoculation  of  the  sterilized  potato  tubes  is  easily 
done.  If  it  is  desired  to  obtain  dilution  cultures,  that  is, 
to  say,  colonies,  this  can  best  be  accomplished  by  making 
several  parallel  streaks  on  the  surface  of  the  potato  with 
the  end  of  a  straight  platinum  wire.  The  same  wire,  un- 
sterilized,  is  then  streaked  repeatedly  over  the  surface  of 
a  second  potato,  and  then  over  that  of  a  third.  The  latter 
will  undoubtedly  have  but  a  few  cells  planted  upon  its  sur- 
face, and,  when  these  multiply,  they  will  yield  isolated 
colonies. 

When  transplanting-  a  pure  culture,  such  as  a  portion 
of  a  colony,  a  single  streak  should  be  made  along  the  mid- 
dle of  the  inclined  potato.  The  characteristics  of  the  re- 
sultant streak  should  be  carefully  noted. 

Laboratory  work. — The  student  will  prepare  20  or  30  potato  tubes 
according-  to  the  directions  given  above.  Streak  cultures  will  be 
made  of  each  of  the  various  organisms  studied. 

Examination  of  Colonies. 


If  the  temperature  is  about  22°  the  gelatin  plates  will 
probably  show  signs  of  growth  in  24  hours.  As  a  rule, 
however,  2  or  3  days  must  be  allowed  for  the  development 
of  colonies.  These  become  visible  to  the  eye,  first  as  mere 
white  points;  eventually,  they  enlarge  and  attain  the  size 
of  a  pin-head,  or  become  even  larger.  A  careful  study  of 
the  colonies  on  the  several  plates  should  first  be  made  with 
the  unaided  eye.  This  macroscopic  examination  is  very  im- 
portant. The  eye  should  be  taught  to  detect  the  first  sign 
of  a  liquefaction,  the  early  appearance  of  pigment,  the 
various  differences  in  the  form,  size,  color  and  structure  of 
the  colonies.  In  this  way  important  information  may  be 
gained  which  will  enable  the  subsequent  work  to  be  done 
to  advantage.  The  various  characteristics  can  often  be 
recognized  at  quite  an  early  period. 


GELATIN   AND    POTATO    MEDIA.  187 

Especial  attention  should  be  given  to  the  form,  and  to 
the  appearance  of  the  colonies;  the  presence,  or  absence  of 
pigment;  liquefaction,  or  non-liquefaction  of  gelatin,  etc. 
It  should  be  remembered,  however,  that  a  given  organism 
may  give  rise  to  at  least  two  kind  of  colonies  which  some 
times  are  quite  different  in  appearance.  Thus,  we  may 
have  surface  as  well  as  deep  colonies.  The  former,  develop- 
ing on  the  surface  of  the  gelatin,  are  unhindered  in  their 
development,  and  may,  therefore,  spread  out  and  thus 
acquire  peculiar  characteristics.  Moreover,  having  ready 
access  to  oxygen,  pigment  formation  and  liquefaction  will 
be  first  seen  in  connection  with  these  surface  colonies. 
The  deep  colonies,  on  the  other  hand,  are  surrounded  on  all 
sides  by  solid  gelatin,  and  hence,  much  the  same  resistance 
to  growth  will  exist  in  all  directions.  The  result  is  that 
the  deep  colonies  of  various  bacteria  may  be  very  much 
alike  in  appearance.  The  deep  colony  is  usually  round  or 
oval,  with  sharp  edges,  and  its  contents  are  slightly  granu- 
lar and  yellowish. 

In  the  study  of  the  various  bacteria,  considerable 
variety  in  the  form  of  the  surface  and  deep  colonies  will  be 
met  with.  In  many  cases  the  growth  is  so  characteristic 
that  it  is  of  great  value  in  the  recognition  of  that  organism. 
It  should  be  remembered,  however,  that  the  cultural  char- 
acteristics of  many  bacteria  are  subject  to  more  or  less 
variation,  depending  on  the  temperature  at  which  the 
growth  occurs,  as  well  as  on  the  reaction  and  consistency 
of  the  gelatin.  The  colonies  on  a  hard,  15  per  cent,  gelatin 
will  be  different,  to  some  extent,  from  those  that  develop 
•on  a  soft,  10  per  cent,  gelatin. 

Plate  1  will  usually  have  a  cloudy  or  milky  appearance. 
On  examination  with  a  low  power — No.  3  objective — the 
cloudiness  will  be  found  to  be  due  to  countless  numbers  of 
minute,  round  colonies.  This  will  be  the*  case  when  the 
material  which  is  employed  for  the  inoculation  is  rich  in 
bacteria.  Only  exceptionally  will  plate  1  have  a  sufficiently 


188  BACTERIOLOGY. 

small  number  of  colonies  to  be  of  value  for  further  study. 
It  is,  therefore,  advisable  to  destroy  this  plate  as  soon  as 
it  has  been  found  to  be  of  no  value.  It  should  be  placed  in 
the  solution  of  mercuric  chloride,  for  at  least  over  night. 

The  object  of  the  plate  method,  it  will  be  remembered, 
is  to  secure  a  perfect  separation  of  the  various  bacteria 
that  may  be  present.  The  farther  apart  these  organisms 
are,  at  the  time  the  gelatin  solidifies,  the  better  it  will  be 
for  subsequent  study.  On  a  good  plate  the  colonies  will  be 
5-10-  mm.  apart.  They  can  be  then  transplanted  without 
any  risk  of  touching  a  neighboring  colony. 

Plate  No.  2  will  sometimes  show  a  successful  dilution. 
This  will  often  be  the  case,  if  but  one  loopful  of  gelatin  is 
transferred.  At  other  times,  the  colonies  on  this  plate 
may  be  so  numerous  as  to  make  it  of  but  little  value. 

Plate  No.  3  should  show  perfectly  distinct  and  well 
separated  colonies.  When  studying  and  transplanting 
these  growths,  care  should  be  taken  not  to  pick  out  an 
accidental  colony  which  owes  its  origin  to  some  organism 
that  dropped  down  from  the  air.  Such  a  foreign  colony  is 
usually  large,  and  grows  rapidly;  moreover,  it  is  likely  to 
be  the  only  one  of  its  kind  on  the  plate.  A  single  large 
colony,  especially  if  unlike  the  others  in  appearance, 
should  always  be  avoided.  This  is  true  when  working  with 
pure  cultures.  When,  however,  some  blood  or  a  portion  of 
an  organ  is  used  for  making  plates  the  single  colony  in  that 
case  may  be  of  great  importance. 

After  having  made  a  careful  study  of  the  various  col- 
onies with  the  unaided  eye,  the  plates  should  then  be  given 
a  careful  microscopic  examination.  The  plate  should  be 
placed  upon  the  stage  of  the  microscope  and  the  colonies 
closely  studied  under  a  low-power,  the  No.  3  objective. 
Further  characteristics  can  thus  be  brought  out  which  have 
escaped  the  eye. 

A  study  of  the  micro-organisms  which  compose  the 
colonies  should  now  be  made.  This  is  done  by  making 


GELATIN  AND    POTATO   MEDIA.  189 

hanging-drop    examinations  and    stained  preparations  ac- 
cording to  the  directions  already  given. 


The  Stab  Culture  in  Gelatin. 

The  object  in  making  plate  cultures  is  to  obtain  colonies 
which,  since  they  are  derived  from  a  single  cell,  are  pure 
cultures  of  that  organism.  To  perpetuate  and  keep  up  the 
pure  culture  thus  obtained,  it  is  necessary  to  resort  to  trans- 
plantation. For  this  purpose,  the  colony  to  be  transplanted 
is  touched  with  a  sterilized  and  cooled,  straight  platinum 
wire.  A  portion  of  the  colony  will  adhere  to  the  end  of  the 
wire,  and  can  be  transferred  to  a  tube  of  sterilized  gelatin. 
The  wire  is  usually  pushed  down  the  center  of  the  solid 
gelatin,  in  which  case  we  have  what  is  known  as  a  stab 
or  stich  culture.  The  operation  of  touching  the  colony 
is  one  that  requires  the  greatest  care  to  prevent  con- 
tamination with  foreign  colonies,  or  with  other  material, 
since  this  would  vitiate  the  pure  culture.  For  this  reason 
it  is  always  carried  out  under  a  microscope,  and  so  far 
as  patience  is  concerned,  it  certainly  is  not  inaptly  called 
"  fishing." 

The  gelatin  plate  is  placed  on  the  stage  of  the  micro- 
scope, and,  with  the  No.  3  objective,  a  suitable  colony  for 
transplantation  is  selected.  It  is  desirable  to  have  but  one 
colony  in  the  field  of  the  microscope.  A  straight  platinum 
wire,  previously  sterilized  and  cooled,  is  held  in  the  right 
hand  in  the  pen  position.  The  hand  is  supported  by  rest- 
ing the  little  finger  on  the  right  corner  of  the  stage.  The 
platinum  wire  is  then  inserted,  about  midway,  between  the 
front  lens  of  the  objective  and  the  surface  of  the  gelatin. 
It  is  held  steadily  in  this  position,  and  on  looking  into 
the  microscope  an  indistinct  shadow  is  seen.  The  wire 
is  slowly  drawn  back  till  the  end  of  the  shadow,  or  in- 
distinct wire  is  directly  over  the  colony.  Should  the  wire 


190  BACTERIOLOGY. 

in  doing  this,  touch  the  objective  or  the  gelatin,  it  must 
be  sterilized  at  once  and  the  operation  repeated.  When 
the  end  of  the  wire  has  been  brought  over  the  colony, 
gradually  lower  the  point  till  it  touches,  or  cuts  the  colony 
into  two. 

Now  carefully  remove  the  wire,  without  touching  the 
microscope  or  some  other  portion  of  the  gelatin.  A  tube  of 
solid  gelatin  is  held  in  the  left  hand  in  an  almost  horizontal 
position,  the  plug  is  then  removed  by  grasping  it  with  the 
little  finger  of  the  right  hand  (Fig.  29).  The  mouth  of  the 
tube  should  be  flamed  in  order  to  remove  any  adherent  cot- 
ton. The  platinum  wire,  which  has  a  small  portion  of  the 
colony  attached  to  it,  is  now  slowly  forced  down  the  center 
of  the  gelatin,  to  the  bottom  of  the  tube.  The  cotton  plug 
is  at  once  replaced,  the  wire  sterilized  and  the  tube  set 
aside. 

Transplantation  can,  of  course,  be  made  direct  from  a 
colony  provided  it  is  very  far  removed  from  any  adjoining 
one.  A  careful  microscopic  examination  should  be  made  of 
the  surrounding  gelatin  looking  out  especially  for  the  pres- 
ence of  exceedingly  small  colonies. 

The  stab  culture  thus  made  is  a  pure  culture,  and  should 
now  be  labelled  with  the  name  of  the  organism  and  date, 
and  set  aside  to  develop.  In  a  few  days  development  takes 
place  along  the  line  of  inoculation,  and,  a  more  or  less  char- 
acteristic growth  results. 

The  manner  of  growth  should  be  observed  daily.  Hang- 
ing-drop and  stained  preparations  can  be  made,  if  it  is  de- 
sired, from  the  tube  cultures.  When  the  gelatin  is  very  old, 
and  hence  too  solid,  it  tends  to  split  as  soon  as  the  plat- 
inum wire  is  forced  into  it.  This  is  remedied  by  melting  the 
gelatin  and  allowing  it  to  re-solidify.  Occasionally,  the 
liquefied  gelatin  is  placed  in  an  inclined  position,  till  it  soli- 
difies. A  streak  culture  can  then  be  made  by  drawing  the  wire 
over  the  surface. 


GELATIN   AND    POTATO   MEDIA.  191 

When  gelatin  tubes,  and  other  plugged  glass-ware  have 
been  kept  for  some  time,  more  or  less  dust  settles  upon  the 
cotton.  There  is,  therefore,  always  danger  of  contamina-_ 
tion  when  removing  a  plug.  This  risk  can  be  reduced  to  a 
minimum,  if  the  cotton  is  touched  to  a  flame  before  it  is  with- 
drawn. It  is  advisable,  as  a  matter  of  routine,  to  scorch 
every  cotton  plug  before  it  is  removed. 

Laboratory  work.— The  student  will  make  a  careful  macroscopic 
and  microscopic  examination  of  the  various  plates.  Stab  cultures 
are  to  be  made  of  each  organism  studied.  Hanging-drop  examina- 
tions and  permanent  stained  preparations  should  be  made,  either  from 
the  colonies  or  from  the  stab  cultures.  The  operation  of  fishing- 
should  be  practised  until  it  can  be  done  readily  and  successfully.  It 
will  be  well  to  have  another  person  watch  the  operation,  to  call  at- 
tention whenever  the  wire  touches  either  the  lens  or  a  part  of  the 
plate,  other  than  that  intended.  Should  the  wire  touch  anything  but 
the  colony  intended,  it  must  be  immediately  sterilized  before  repeat- 
ing the  attempt. 

The  line  of  study  of  the  Bacillus  prodigiosus,  Bacillus 
Indicus  and  of  the  various  bacteria  to  be  presently  taken 
up  consists,  first,  in  making  plate  cultures.  Colonies  are 
thus  obtained,  the  characteristics  of  which  are  thoroughly 
studied.  Hanging-drop  examinations,  and  stained  prepara- 
tions are  next  made,  in  order  to  become  familiar  with  the 
organism  itself.  Stab  cultures  in  gelatin  are  then  made, 
and  also  streak  cultures  on  potato.  Later  on,  when  the 
study  of  the  pathogenic  organisms  is  taken  up,  cultures 
are  also  made  on  agar,  in  bouillon  and  in  milk.  Finally 
drawings  are  made  showing  the  form  of  the  colony,  the 
form  of  the  organism,  the  appearance  of  the  stab  culture, 
etc. 

In  order  to  economize  on  gelatin,  one-half  of  the  class 
makes  plates  of  one  set  of  organisms,  and,  the  next  timer 
the  other  half  of  the  class  does  the  plating.  Each  student, 
however,  is  expected  to  examine  and  study  every  organism 
which  is  given  out. 


192  BACTERIOLOGY. 

Summarized,  then,  each  organism  is  to  be  studied  by 
making-: 

Plates, 

Colonies, 

Hanging-drop  examination, 
Stained  preparation, 
Stab  culture  in  gelatin, 
Streak  culture  on  potato,  or  agar,  or  both. 
Drawings. 

The  laboratory  work,  during-  the  first  few  weeks,  is  carried  on  as 
nearly  as  possible  according-  to  the  following  schedule: 

1st  day.— Cleaning-  up,  and  plugging  of  tubes. 

2nd  day. — Preparation  of  gelatin;  sterilization  and  filling  of 
tubes. 

3rd  day. — Dilution  and  mass  potato  cultures;  sterilization  of 
gelatin. 

4th  day. — Sterilization  of  gelatin;  use  of  microscope;  hanging- 
drop  work. 

5th  day. — Hanging-drop  work,  continued;  simple  staining. 

6th  day.— Gelatin  plates  (prodigiosus  and  IndicusJ;  simple  stain- 
ing, continued. 

7th  day. — Study  of  colonies,  stab  cultures,  etc. 

8th  day.— Gelatin  plates  (red  and  violet  bacilli);  Petri  dishes 
(orange  sarcine);  Esmarch  rolls  (yellow  sarcine). 

9th  day.— Study  of  colonies,  stab  cultures,  etc. 
10th  day.— Gelatin  plates  (Kiel  and  fluorescing  bacilli);  Esmarch 
dilution  potato  (prodigiosus);  potato  tube  culture. 

The  plating  of  two  organisms,  on  alternate  days,  is  continued 
till  the  non-pathogenic  bacteria  have  been  covered.  The  class  then 
takes  up  the  work  on  yeasts  and  moulds,  as  given  in  chapter  XII. 
This  is  followed  by  the  examination  of  air,  water  and  soil,  according 
to  the  directions  laid  down  in  chapter  XIII.  The  preparation  of 
bouillon  and  agar  completes  the  first  half  of  the  course. 

The  class  then  enters  upon  the  study  of  pathogenic  bacteria,  be- 
ginning with  the  work  on  anthrax  animals.  The  study  of  the  anae- 
robic bacteria  is  followed  by  that  of  the  remaining  organisms.  These 
are  supplied  as  pure  cultures,  or  in  infected  animals. 


CHAPTER  VIII. 
THE  NON-PATHOGENIC  BACTERIA. 


BACILLUS  PRODIGIOSUS.— B.  INDICUS.— B.  RUBER  OF  KIEL.— B.  RUBI- 

DUS.— B.  VIOLACEUS.— B.  FLUORESCENS  PUTIDUS.— B.  PHOSPHOR- 

ESCENS.— ORANGE  SARCINE.— YELLOW  SARCINE.— B.  SUB- 

TILIS. — B.     MESENTERICUS    VULGATUS. — B.     MEGA- 

TERIUM. — B.  RAMOSUS. — PROTEUS  VULGARIS. — 

BACTERIUM  ZOPFII. — SPIRILLUM  RUBRUM. 

— B.     ACIDI    LACTIC!.— B.     BUTYR- 
ICUS.—  B.       CYANOGENUS. 


193 

13 


Bacillus   Prodigiosus. 

MONAS  PRODIGIOSA,   of  Ehrenberg.      M1CROCOCCUS  PRODIGIOSUS. 

ORIGIN. — Pound  on  starchy  substances,  rice,  potatoes, 
moist  bread;  also  on  meat,  albumin,  milk,  etc.  May  cause 
at  times  local  "epidemics,"  infecting-  foods  as  bread,  meat, 
and  sausages,  which  assume  a  pink  or  red  color.  "Bleed- 
ing1" bread  or  wafers  (p.  116). 

FORM  — A  very  short  rod,  slightly  longer  than  its  width. 
May  form  short  threads,  especially  in  old  cultures  or  in 
slightly  acid  media.  Usually  single  or  in  pairs. 

MOTILITY. — Ordinarily  shows  no  motion  other  than  a 
marked  Brownian  movement.  In  acid  or  very  dilute  media 
the  slimy  character  of  the  growth  decreases  and,  as  a 
result,  a  slight  motion  is  observed;  whips  have  been 
demonstrated. 

SPORULATION. — Has  not  been  observed.  It  possesses, 
however,  marked  resistance  to  desiccation. 

ANILIN  DYES.' — Stain  readily. 

GROWTH. — Very  rapid. 

Gelatin  plates.— Deep  colonies,  round  or  oval,  with  sharp  border 
and  light  brown  color.  Surface  colonies,  irregular,  rough  border, 
granular,  with  reddish  center,  and  surrounded  by  clear,  liquefied 
gelatin. 

Stab  culture.—  Rapid,  funnel-shaped  liquefaction,  extending  along 
the  entire  line  of  inoculation.  A  red  scum  forms  on  the  surface 
of  the  liquid;  this  eventually  settles  and  the  entire  contents  of  the 
tube  are  colored  bright  red. 

Streak  culture. — On  agar,  it  forms  an  abundant,  moist,  spreading 
growth,  having  an  intense  red  color  which  is  non-diffusible.  On.  potato. 
the  growth  is  especially  rapid,  and  slimy,  with  marked  pigment  pro- 
duction. The  pigment,  when  old,  has  a  metallic,  fuchsin-like  luster, 
Odor  of  trimethylamin.  On  blood-serum,  growth  as  on  agar,  with  liq- 
uefaction. 

Milk. — Growth  takes  place  and  the  pigment  is  held  in  solution  by 
the  fat  globules.  Coagulation  results. 

OXYGEN  REQUIREMENTS. — It  is  a  facultative  anaerobe. 

TEMPERATURE. — Grows  best  at  ordinary  room  temperature.  In 
the  incubator  it  ceases  to  form  pigment,  and  may  temporarily  lose 
this  property,  i.  e.,  becomes  attenuated. 

BEHAVIOR  TO  GELATIN. — Rapidly  liquefies  as  the  result  of  the 
formation  of  a  soluble  peptonizing  ferment.  This  liquefying  property 
may  be  diminished,  or  temporarily  lost  by  growth  in  acid  media. 

AEROGENESIS. — Strong  odor  of  trimethylamin  on  potatoes.  It 
ferments  sugar  solutions. 

PIGMENT  PRODUCTION. — On  various  media  a  bright  red  pigment 
forms.  This  is  soluble  in  alcohol,  ether,  chloroform,  etc.  It  is  formed 
only  in  the  presence  of  air,  and  at  ordinary  temperature;  not  at  37°. 

PATHOGENESIS. — It  is  not  pathogenic.  Its  soluble  products  in 
large  amounts  may  have  a  toxic  action.  The  cellular  proteins  may 
induce  suppuration.  Animals  insusceptible  to  malignant  edema  are 
rendered  susceptible  by  an  injection  of  this  bacillus.  Rabbits  inocu- 
lated with  anthrax  are  saved  by  the  injection  of  Bacillus  prodigiosus. 

194 


DRAWINGS.  195 


Bacillus  Indicus,  Koch. 

B.    RUBER  INDICUS. 

ORIGIN.— Isolated  in  India  from  the  contents  of  the 
stomach  of  a  monkey. 

FORM. — Small,  narrow,  very  short  rod  with  rounded  ends. 
MOTILITY. — Actively  motile. 
SPORULATION. — Not  definitely  observed. 
ANILIN  DYES. — Stain  readily. 
GROWTH. — Is  rapid. 

Gelatin  plates.—  Deep  colonies  are  yellowish,  with  a  wavy  contour. 
Surface  colonies  grayish  yellow,  finely  granular,  with  fibrillated  bor- 
ders. Show  movement  of  contents,  rapidly  liquefy  and  may  show  a 
lig-ht  pink  color. 

Stab  culture. — Rapid  liquefaction  along"  line  of  inoculation. 
Dense  flocculent  growth  settles  on  the  bottom,  and  is  grayish  or  light 
pink  in  color.  A  delicate  scum  forms  on  the  surface  and  is  colored 
from  a  light  pink  to  a  brick  red. 

Streak  culture.  -  On  agar,  it  forms  a  low,  moist,  spreading"  growth 
which  usually  is  faint  pink  in  color.  On  potato,  the  growth  is  low, 
not  slimy  as  that  of  B.  prodigiosus,  and  the  color  is  more  marked 
than  on  other  media.  On  blood-serum,  liquefaction  results  with,  or 
without  pigment  production. 

OXYGEN  REQUIREMENTS. — It  grows  best  in  the  presence  of 
air,  but  is  a  facultative  anaerobe.  The  pigment  production 
depends  upon  the  presence  of  oxygen. 

TEMPERATURE. — The  optimum  is  about  35°.  The  pig- 
ment is  absent  in  cultures  that  develop  in  the  incubator. 
Splendid  pigment  when  growth  occurs  at  15°. 

BEHAVIOR  TO  GELATIN. — It  liquefies  very  rapidly. 

PIGMENT  PRODUCTION. — Varies  greatly.  The  pigment 
may  be  grayish  to  bright  brick-red;  lacks  the  violet  tinge 
•of  B.  prodigiosus.  Usually  it  is  light  pink,  so  that  the 
ordinary  cultures  may  be  considered  as  attenuated. 

PATHOGENESIS. — It  has  a  marked  toxic  action,  and  when 
Injected  in  large  amounts  into  the  abdominal  cavity,  or 
into  the  veins  of  rabbits  and  guinea-pigs,  it  proves  fatal. 
Rabbits  develop  marked  diarrhea  and  die  in  from  3  to  20 
hours.  On  post-mortem  the  intestines  show  a  severe  inflam- 
matory condition  of  the  mucous  membrane;  at  times, 
ulcerations  are  present. 

196 


DRAWINGS.  197 


Bacillus  Ruber  of  Kiel,  Breunig. 

B.    RUBER  BALTICUS. 

ORIGIN. — Drinking  water  of  Kiel. 

FORM. — Thick  rods  two  to  three  times  as  long  as  wide; 
at  times,  may  be  much  longer. 

MOTILITY.  — It  is  somewhat  motile,  and  the  motion  de- 
pends on  presence  of  oxygen. 

SPORULATION. — This  has  not  been  observed. 

ANILIN  DYES. — Stain  readily. 

GROWTH. — Rapid  and  abundant. 

Gelatin  plates. — Deep  colonies  are  oval,  pale  yellow,  with  wavy  or 
even  border.  The  surface  colonies  are  blood-red  in  color,  spread 
rapidly,  and  have  a  sinuous  border;  are  surrounded  by  a  clear  zone, 
and  liquefy  gelatin.  £Z 

Stab  culture. — Growth  and  liquefaction  take  place  along*  the  line 
of  inoculation.  The  fluid  becomes  strongly  colored,  and  gas  may 
form  in  the  deeper  layers. 

Streak  culture. — On  agar,  at  30-35°,  the  thick,  slimy  growth  is  at 
first  a  pale  rose,  and  later  becomes  a  brick-red.  On  potato,  at  30-35°, 
it  develops  rapidly,  forming-  a  purple  red  growth.  At  lower  tem- 
peratures the  color  is  very  marked-;  at  first  it  is  orange,  later  carmine 
red. 

Milk. — At  35°,  coagulation  takes  place  in  24  hours,  without  a 
trace  of  coloration,  due  to  rapid  growth  and  production  of  acidity. 
At  ordinary  temperature  the  coagulation  takes  place  slowly,  and  the 
fluid  gradually  colors. 

OXYGEN  REQUIREMENTS. — It  is  a  facultative  anaerobe,  but 
requires  oxygen  to  form  the  pigment. 

TEMPERATURE. — Grows  from  10  to  42°.  The  optimum  is 
30-35°,  and  above  this  the  growth  ceases  to  be  colored. 
Direct  insolation  kills  it  in  five  hours.  An  exposure  of 
three  hours  does  not  kill,  but  alters  it  so  that  it  no  longer 
produces  the  pigment,  i.  e. ,  becomes  attenuated. 

BEHAVIOR  TO  GELATIN. — It  liquefies  gelatin  quite  rapidly. 

AEROGENESIS. — Gas   bubbles   form   in    the   gelatin. 

PIGMENT  PRODUCTION.— This  weakens  considerably  on  cultiva- 
tion, and  almost  colorless  varieties  are  met  with.  The  color  may  be 
restored  by  successive  culture  on  potato,  and  by  growth  at  15°.  It  is 
soluble  in  alcohol,  but  not  in  chloroform.  The  pigment  is  turned 
red  by  acids,  and  is  decolored  by  alkalis.  It  is  discolored  by  zinc  dust 
and  glacial  acetic  acid— distinction  from  that  of  B.  prodigiosus. 

198 


DRAWINGS.  199 


Bacillus  Rubidus. 

B.   RUBER  BEROLINENSIS. 

ORIGIN. — Water  supply  of  Berlin. 
FORM. — Long1,  narrow  rod;  forms  threads. 
MOTILITY. — Actively  motile. 
SPORULATION. — No  spores  observed. 
ANILIN  DYES. — Stain  readily. 
GROWTH. — Fairly  rapid. 

Gelatin  plates. — Small,  yellow,  finely  granular  colonies,  with  irreg- 
ular border.  It  liquefies  gelatin. 

Stab  culture.—  Gelatin  slowly  liquefies  along-  line  of  inoculation. 
A  yellowish  mass  of  bacteria  settles  to  the  bottom,  and  a  thin  folded 
scum  forms  on  the  surface. 

Streak  culture. — On  agar,  it  forms  a  thin,  irregularly  bordered, 
slightly  folded,  yellowish  growth.  On  potato,  the  most  characteristic 
growth  forms;  it  spreads,  and  has  a  bright  brick-red  color.  On  blood- 
serum,  liquefaction  takes  place  and  a  red  pigment  forms. 

OXYGEN  REQUIREMENTS. — It  is  aerobic. 
TEMPERATURE. — It  does  not  grow  at  the  temperature  of 
the  body. 

BEHAVIOR  TO  GELATIN. — Liquefies. 
PATHOGENESIS. — No  effect  observed. 

An  interesting-  red  pigment-producing  bacillus,  allied 
to  the  B.  prodigiosus  and  to  the  Kiel  bacillus,  has  been 
found  on  freshly  packed  oil  sardines,  and  also  in  the  sup- 
purating felons  on  the  fingers  of  the  packers.  In  the  lat- 
ter case  it  was  associated  with  an  anaerobe. 

About  ten  additional  species  or  varieties  of  bacilli 
are  known,  that  are  characterized  by  the  production  of  a 
red  pigment.  Moreover,  a  red  pigment  can  be  produced  by 

various  micrococci,  sarcines,  spirilla  and  yeasts. 

200 


DRAWINGS.  201 


Bacillus  Violaceus. 

B.    VIOLACEUS  BEROLINENSIS.      VIOLET  BACILLUS  OF   WATER. 

ORIGIN. — Water  of  the  river  Spree  at  Berlin,  and  of  the 
Thames  at  London;  also  in  well  water. 

FORM. — A  slender  rod  about  two  to  three  times  as  long 
as  wide;  it  forms  threads,  but  is  usually  in  pairs. 

MOTILITY. — It  is  actively  motile. 

SPORULATION. — Forms  median  spores. 

ANILIN  DYES. — React  readily. 

GROWTH. — This  is  moderately  rapid. 

Gelatin  plates. — The  colony  is  irregular  with  loose  fibrillated  bor- 
der. The  center  shows  quite  early  a  violet  color.  It  liquefies. 

Stab  culture. — Funnel-shaped  liquefaction  along  the  entire  line  of 
inoculation.  A  violet  sediment  collects  on  the  bottom,  while  the 
liquefied  gelatin  above  is  perfectly  clear.  A  violet  ring  may  adhere 
to  the  wall  of  the  tube  on  the  surface  of  the  liquid. 

Streak  culture. — On  agar,  it  forms  a  smooth,  thin,  moist,  bright  vio- 
let covering.  On  potato,  the  growth  is  somewhat  slow  but  very  char- 
acteristic, forming  a  bright  violet,  eventually  dark  covering.  On 
blood-serum,  a  violet  color  forms  and  liquefaction  takes  place. 

OXYGEN  REQUIREMENTS. — It  is  a  facultative  anaerobe. 
Oxygen  is  necessary  to  pigment  formation. 

TEMPERATURE.  —It  does  not  grow  at  higher  temperatures. 

BEHAVIOR  TO  GELATIN. — Liquefies. 

PIGMENT  PRODUCTION. — This  depends  upon  the  presence 
of  oxygen.  The  color  is  soluble  in  alcohol,  insoluble  in 
ether  and  in  chloroform.  It  is  changed  to  a  green  by  min- 
eral acids. 

PATHOGENESIS. — It  has  no  effect. 

About  ten  additional  violet  or  blue  pigment  producing 
bacilli  are  known.  Some  of  these,  however,  may  be  mere 

varieties. 

202 


DRAWINGS. 


Bacillus  Fluorescens   Putidus,   Flugge. 

FLUORESCING  BACILLUS  OF  WATER. 

.ORIGIN. — Putrid  media,  water. 
FORM. — Short,  small  rods,  with  rounded  ends. 
MOTILITY. — Very  actively  motile. 

SPORULATION. — No  spores  observed  on  ordinary  media,. 
On  althea,  or  on  quince-jelly  splendid  spores  form  (Migula). 
ANILIN  DYES. — Stain  readily. 
GROWTH.  — Rapid. 

Gelatin  plates.— The  deep  colonies  are  small,  round,  and  finely 
granular.  The  surface  colonies  spread  rapidly  and  form  at  first  a 
very  thin  plaque,  with  irregular,  wavy  border  which  shows  markings. 
Later,  a  bluish  green  color  diffuses  through  the  surrounding  gelatin. 
Odor  of  trimethylamin.  No  liquefaction. 

Stab  culture.— No  growth  in  the  lower  part  of  the  tube.  The  sur- 
face of  the  gelatin  is  covered  with  a  grayish  white  growth.  The 
fluorescing  pigment  gradually  diffuses  downward  into  the  gelatin. 
The  gelatin  itself  becomes  yellowish. 

Streak  culture.— On  agar,  a  moist,  spreading  growth.  The  agar  be- 
comes greenish,  but  later  on  the  color  fades.  On  potato,  a  thin  gray- 
ish or  brownish,  moist  growth  forms. 

OXYGEN  REQUIREMENTS. — Aerobic. 

TEMPERATURE. — Ordinary  room  temperature  is  best. 

BEHAVIOR  TO  GELATIN. — It  does  not  liquefy. 

PIGMENT  PRODUCTION. — This  occurs  only  in  the  presence- 
of  air.  Sulphur  and  phosphorus  seem  to  be  necessary. 
The  coloring  matter  is  soluble  in  water,  and  hence, 
diffuses  into  the  surrounding  medium.  It  possesses  fluor- 
escing properties;  in  other  words  it  exhibits  one  color  by 
transmitted  light  (yellow),  and  another  (bluish-green),  by 
reflected  light.  The  pigment  is  destroyed  by  acids  and  is 
favored  by  alkali,  such  as  ammonia.  Colorless  varieties  can 
be  obtained,  by  insolation  and  otherwise,  as  in  the  case  of 
other  pigment  bacteria. 

PATHOGENESIS.— It  is  without  action  on  animals. 

About  a  score  of  species  or  varieties  of  liquefying  and 
non-liquefying  fluorescing  bacteria  have  been  isolated  from 
water,  air,  soil  and  various  sources.  The  green  diarrhea 
of  children,  green  sputum,  etc.,  may  in  some  cases,  be  due 
to  organisms  of  this  class. 

204 


DRAWINGS.  205 


Bacterium  Phosphorescens,  Fischer. 

PHOTOBACTERIUM  PHOSPHORESCENS. 

ORIGIN. — Pound  in  the  water  of  the  harbor  of  Kiel,  also- 
on  dead  sea-fish,  oysters,  etc.  From  these  it  may  spread  to 
meat  in  butcher  shops. 

FORM. — Short,  thick  bacillus,  with  rounded  ends;  some- 
times almost  a  coccus.  It  is  usually  in  pairs,  but  may  form 
threads.  Involution  forms  soon  develop. 

MOTILITY. — No  motion. 

SPORULATION. — This  has  not  been  observed. 

ANILIN  DYES. — Stain  readily;     so  does  Gram's  method. 

GROWTH. — Is  moderately  rapid.  The  cultures  show  in 
the  dark  a  marked  bluish-green  phosphorescence  (see  p.  116). 

Gelatin  plates.— Show  small,  white,  glistening-  colonies,  which  do- 
not  liquefy.  The  border  is  sharp,  irregular,  and  the  contents  are 
granular,  and  show  several  concentric  ring's. 

Stab  culture. — A  slight  granular  growth  along"  the  line  of  inocu- 
lation. Most  abundant  on  the  surface,  where  it  forms  forms  a  thin 
grayish  white  covering-.  Eventually,  the  gelatin  is  colored  a  yellow- 
ish brown. 

Streak  culture. — On  aqar,  potato,  etc.,  growth  is  limited  to  the 
line  of  inoculation.  It  grows  well  on  fish,  beef,  bread,  fats,  etc. 

OXYGEN  REQUIREMENTS. — It  is  a  facultative  anaerobe. 
The  production  of  light  depends  upon  the  presence  of  oxy- 
gen, and  is,  therefore,  most  marked  on  the  surface  growths. 
The  intensity  of  the  light  may  diminish,  and,  may  become 
lost — attenuation.  It  may  be  restored  by  growth  on  suita- 
ble media,  such  as  fish.1 

TEMPERATURE. — Does  not  grow  in  the  incubator,  but  may 
grow  at  0°. 

BEHAVIOR  TO  GELATIN. — It  does  not  liquefy  gelatin,  but 
can  ferment  sugar. 

PATHOGENESIS. — No  effect  on  animals.  One  phosphores- 
cing bacillus  is  said  to  produce  a  disease  in  certain  crus- 
tacea. 

1  A  good  medium  for  the  growth  of  these  organisms  is  prepared 
in  the  same  manner  as  ordinary  gelatin,  by  adding  500  g.  of  chopped 
fish  to  1,000  c.c.  of  water.  The  material  is  digested,  and  strained. 
To  the  filtrate  100  g.  of  gelatin,  40  g.  of  salt,  5  g.  of  glycerin,  and 
5  g.  of  asparagin  are  added,  and  the  mixture  is  rendered  slightly 
alkaline  (p.  155).  The  liquid  after  heating  is  filtered,  placed  in  tubes 
and  sterilized. 

206 


DRAWINGS.  20T 


Sarcina  Aurantiaca. 

ORANGE  SARCINE. 

ORIGIN. — From  air,  also  from  weiss-beer. 

FORM. — Small,  spherical  cocci,  grouped  as  diplococci 
or  tetrads,  and  at  times  in  package-shaped  masses.  The 
latter  are  especially  constant  in  hay  infusions. 

MOTILITY — None. 

SPORULATION. — None. 

ANILIN  DYES. — Stain  very  easily,  and  are  likely  to 
overstain. 

GROWTH. — Rather  rapid. 

Gelatin  plates. — Show  round,  sharp-edged  colonies,  which  are 
granular  and  of  an  orange-yellow  color.  These  soon  liquefy  the 
gelatin. 

Stab  culture. — The  gelatin  is  liquefied  along  the  entire  line  of 
inoculation.  Eventually,  an  orange-colored  deposit  of  bacteria  forms 
on  the  bottom,  and  the  liquid  above  becomes  clear. 

Streak  culture. — On  agar,  it  forms  a  thick,  orange-colored  growth. 
On  potato,  the  pigment  is  excellently  developed,  and  the  growth  is 
thick. 

OXYGEN  REQUIREMENTS. — It  is  aerobic. 
TEMPERATURE. — A  high  temperature  is  unfavorable. 
BEHAVIOR  TO  GELATIN. — It  liquefies  rapidly. 
AEROGENESIS. — Not  observed 
PATHOGENESIS. — It  has  no  effect  on  animals. 

A  red  sarcine  has  been  found  in  so-called  "red  milk" 
—a  condition  which  at  times  may  be  due  to  B.  prodigiosiis, 
or  to  red  yeasts.  A  somewhat  similar  micrococcus  has 
been  found  in  "red  sweat."  It  is  interesting- to  note  that 
the  sarcine  commonly  found  in  the  lungs  is  said  to  form 
spores.  Another  red  sarcine,  isolated  from  an  ascitic  fluid, 
possessed  active  motion.  A  motile  sarcine  with  giant- 
whips  has  been  observed. 

208 


DRAWINGS.  209 


14 


Sarcina  Lutea,  Schroter. 

YELLOW  SARCINE. 

ORIGIN. — Air. 

FORM. — Larger  cocci  than  the  orange  sarcine,  and 
moreover,  it  forms  more  perfect  package-shaped  masses. 

MOTILITY.  — None. 

SPORULATION  .  — None. 

ANILIN  DYES. — React  readily  and  are  likely  to  over- 
stain.  The  characteristic  division  of  the  cells  is  then  lost. 

GROWTH. — Very  slow. 

Gelatin  plates. — Colonies  develop  very  slowly  as  minute  yellowish 
spots,  which  show  an  irregular  oblong-  form  and  are  markedly  granular. 
The  colonies  do  not  liquefy  g-elatin. 

Stab  culture.— Growth  is  especially  developed  on  the  surface  and 
extends  but  slightly  down  the  line  of  inoculation.  Lower  half  of 
tube  is  usually  free  from  growth,  or  at  most,  contains  a  few,  isolated, 
spherical  colonies.  The  color  is  brig-ht  yellow  and  in  very  old  tubes 
liquefaction  slowly  shows  itself,  so  that  eventually  a  brig-lit  yellow 
deposit  forms  on  the  bottom  while  the  supernatant  liquefied  g-elatin 
is  perfectly  clear. 

Streak  culture. — On  agar,  it  forms  a  very  thick,  moist,  brig-ht 
yellow  covering-.  On  potato,  the  growth  is  slow, with  production  of 
the  same  thick  growth  and  color. 

OXYGEN  REQUIREMENTS. — It  is  an  aerobic  organism. 
TEMPERATURE. — It  may  grow  in  the  incubator. 
BEHAVIOR  TO  GELATIN. — A  very  slow  liquefaction  is  ob- 
served after  a  lapse  of  several  weeks. 
PATHOGENESIS. — None. 

The  sarcine  form  was  first  observed  in  1842  in  vomited 
stomach  contents  and  was  designated  as  S.  ventriculi. 
Within  recent  years  a  stomach  sarcine  has  been  isolated  in 
pure  culture  and  found  to  correspond,  in  many  respects,  to 
the  yellow  sarcine.  More  than  one  species,  however,  may 

exist  in  the  contents  of  the  stomach. 

210 


DRAWINGS.  211 


Bacillus  Subtilis,  Ehrenberg. 

HAY  BACILLUS. 

ORIGIN. — In  air,  water,  soil,  feces,  putrid  fluids,  and  in 
infusions  of  hay. 

FORM. — Large,  rather  thick  rods,  3-4  times  as  long- 
as  wide,  with  rounded  ends.  Usually  in  pairs,  often  in  threads. 

MOTILITY. -Active  snake-like  motion.  8-12  lateral  flagella. 

SPORULATION. — It  forms  large,  oval  spores  at  or  near 
the  middle,  without  enlargement.  They  are  highly  resist- 
ant and  can  be  readily  double  stained.  Germination,  p.  54. 

ANILIN  DYES. — Stain  readily;  so  does  Gram's  method. 

GROWTH. — Very  rapid.  At  21°  cell  division  has  been 
observed  to  take  place  in  75  min. ;  and  at  35°  in  20  min. 

GelaMn  plates.— The  surface  colonies  liquefy  gelatin  rapidly  and 
extensively,  and  present  a  striking-  appearance.  The  central  portion 
appears  as  a  grayish  yellow,  irregular  mass,  which  on  close  examina- 
tion can  be  seen  to  be  made  up  of  moving  cells.  This  central  portion 
is  surrounded  by  a  lighter,  granular  zone.  The  border  of  the  colony  is 
quite  characteristic.  It  consists  of  a  dense  zone  of  bacilli  and 
threads,  radially  arranged,  so  that  the  ends  project  outward,  thus 
presenting  a  peculiar  appearance — the  so-called  "ray  crown." 

Stab  culture.— Very  rapid,  funnel-shaped  liquefaction  takes  place 
along  the  entire  line  of  inoculation.  White  flocculent  masses  ac- 
cumulate at  the  bottom  while  the  liquid  above,  at  first  turbid,  becomes 
clear.  On  the  surface  a  dense  white  scum  or  zooglea  usually  forms. 

Streak  culture.— On  agar,  it  forms  a  dull  grayish  white,  thick, 
folded  scum.  On  potato,  it  develops  excellently,  and  forms  a  moist, 
thick,  yellowish-white  covering,  which  at  first  is  velvety  in  appear- 
ance but  later  becomes  dry  and  granular,  and  contains  spores  as  well 
as  involution  forms.  On  blood-serum,  it  also  forms  a  folded  scum  and 
liquefies. 

OXYGEN  REQUIREMENTS. — It  is  aerobic. 

TEMPERATURE. — Grows  from  10  to  45°.   Optimum  about  30°. 

BEHAVIOR  TO  GELATIN. -It  liquefies  rapidly  and  extensively. 

PATHOGENESIS. — It  has  no  pathogenic  power.  When 
a  large  number  of  spores,  are  injected  into  the  blood  they 
,soon  disappear  and  are  taken  up  by  the  liver  and  spleen. 
They  may  be  stored  up  in  these  organs  for  60  to  70  days 
and  yet  preserve  their  vitality  (Wyssokowitsch). 

A  large  number  of  bacilli  resemble  the  above  B.  subtilis 
to  a  marked  extent.  It  is  customary,  therefore,  to  speak 
of  the  group  of  hay  bacilli. 

212 


DRAWINGS.  213 


Bacillus  Mesentericus  Vulgatus,  Fliigge. 

POTATO  BACILLUS. 

ORIGIN.  -Widely  distributed  in  the  soil,  on  the  surface 
of  potatoes,  in  feces,  putrid  fluids,  water,  milk,  etc. 

FORM.— Small,  thick  rods,  with  rounded  ends,  usually 
in  pairs,  may  form  threads. 

MOTILITY. — Actively  motile;  flagella  numerous. 

SPORULATION. — It  readily  forms  large  median,  round- 
ish spores.  The  spores  of  one  variety  of  "potato  bacillus" 
described  by  Globig,  showed  enormous  powers  of  resistance, 
withstanding-  the  action  of  steam-heat  for  five  to  six  hours. 

ANILIN  DYES. — React  easily;  so  does  Gram's  method. 

GROWTH. — Is  very  rapid,  and  in  many  respects  resem- 
bles that  of  the  hay  bacillus. 

Gelatin  plates. — Show  yellowish  white,  slightly  granular  colonies, 
with  irregular  borders.  They  liquefy  rapidly  and  extensively. 

Stab  culture.—  Growth  occurs  along  the  entire  line  of  inoculation, 
but  liquefaction  is  more  energetic  in  the  upper  part.  The  liquefied 
gelatin  remains  turbid  for  some  time  and  a  thin,  grayish,  folded  scum 
forms  on  the  top. 

Streak  culture. — On  agar,  it  forms  a  dull  white  or  grayish,  folded 
growth.  On  potato,  the  most  characteristic  growth  develops.  The 
surface  is  rapidly  covered  with  a  thick,  white,  strongly  folded,  coher- 
ent growth.  Later,  the  growth  becomes  dirty  brown  or  red  in  color. 

MILK.— Casein  is  coagulated,  and peptonized.  Starch  is 
inverted. 

OXYGEN  REQUIREMENTS. — It  is  aerobic. 

TEMPERATURE. — It  grows  at  ordinary,  as  well  as  at 
higher  temperatures. 

BEHAVIOR  TO  GELATIN. — It  liquefies  rapidly. 

PATHOGENESIS. — No  effect  observed. 

Several  varieties  of  "potato  bacilli"  have  been  met 
with.  Some  form  a  brown,  while  others  give  a  reddish 
growth  on  potatoes.  The  spores  of  the  potato  and  hay 
bacilli  are  extremely  resistant.  When  the  material  is  in  a 
small  mass,  not  in  a  fine  state  of  suspension,  it  may  re- 
quire an  exposure  to  steam  of  10  hours  or  more,  in, order  to 

insure  sterilization  (p.  162). 

214 


DRAWINGS.  215 


Bacillus  Megaterium,  De  Bary. 

ORIGIN. — Originally  found  on  boiled  cabbage  leaves, 
but  may  be  present  on  other  vegetable  matter;  also  found 
in  the  air  and  in  soil. 

FORM. — Large  cylindrical  rods,  with  granular  contents, 
three  to  six  times  as  long  as  broad,  with  rounded  ends. 
They  are  usually  slightly  bent,  are  in  pairs,  and  may  form 
threads.  Involution  forms  are  very  common.  Capsulated 
cells  are  especially  found  in  the  slimy  growths. 

MOTILITY. — Slow,  creeping  motion.     6-8  lateral  flagella. 

SPORULATION. —  Worms  median  spores. 

ANILIN  DYES. — Stain  readily,  though  irregularities  due 
to  granular  protoplasm  may  be  seen. 

GROWTH.  — Rapid. 

Gelatin  plates. — Colonies  are  at  first  irregular,  small,  yellowish 
masses,  but  subsequently  show  marked  radiating  or  branching-  forms, 
which  soon  liquefy  the  gelatin.  Kidney-shaped  colonies  may  be  met 
with. 

Stab  culture. — Rapid  growth  and  liquefaction  along  the  line  of 
inoculation.  May  show  threads  of  bacteria  penetrating  outward  into 
the  solid  gelatin.  Eventually,  the  gelatin  is  wholly  liquefied  and  a 
flocculent  mass  accumulates  on  the  bottom;  the  supernatant  liquid 
clears  up  without  formation  of  scum  on  top. 

Streak  culture. — On  agar,  it  forms  a  dull  white  or  grayish  cover- 
ing. On  potato,  it  grows  rapidly  a?  a  thick,  slimy,  grayish  white 
mass,  which  is  rich  in  spores  and  involution  forms. 

OXYGEN  REQUIREMENTS. — It  is  aerobic. 
TEMPERATURE. — The  optimum  is  at  about  20°.    It  may, 
however,  grow  in  the  incubator. 

BEHAVIOR  TO  GELATIN. — It  liquefies  rather  slowly. 

PATHOGENESIS. — No  effect  observed. 

216 


DRAWINGS.  21T 


Bacillus  Ramosus. 
ROOT  OR  "WURZEL"  BACILLUS. 

ORIGIN. — Very  common  in  earth;  occurs  also  in  river 
and  in  spring  water. 

FORM.  —Rather  large  rods,  thicker  than  the  Hay  bacil- 
lus; with  slightly  rounded  ends.  Threads  are  common. 

MOTILITY. — It  is  slowly  motile. 

SPORULATION. — Large  median  spores  occur. 

ANILIN  DYES. — It  stains  well. 

GROWTH.— Rapid. 

Gelatin  plates. — The  colonies  present  a  characteristic  appearance, 
resembling-  somewhat  fine  branching-  rootlets,  hence  the  name.  At 
first  the  colonies  are  round,  dark  and  with  bristly  borders.  Subse- 
quently the  colonies  branch  and  ramify  throughout  the  gelatin  which 
is  slowly  liquefied. 

Stab  culture. — This  is  also  characteristic.  Growth  develops  along- 
the  line  of  inoculation  and  from  this  threads  penetrate  or  radiate  into 
the  surrounding-  g-elatin.  The  growth  is  more  rapid  at  the  top  than  in 
the  lower  parts  of  the  tube  so  that  the  appearance  of  an  "inverted  pine 
tree  "  results.  Later  the  gelatin  is  liquefied  completely.  The  bacter- 
ial growth  accumulates  on  the  bottom  while  the  liquid  above  becomes 
clear  and  has  a  thin  scum  on  the  surface. 

Streak  culture. — On  agar  it  forms  a  grayish  growth,  spreading-  out- 
ward from  the  streak  so  that  the  appearance  often  is  not  unlike  that 
of  a  centipede.  On  potato,  a  slimy,  whitish  growth  develops,  which 
is  rich  in  spores. 

OXYGEN  REQUIREMENTS. — It  is  aerobic. 
TEMPERATURE. — It  grows  at  ordinary  temperature,  and 
.also  in  the  incubator. 

BEHAVIOR  TO  GELATIN. — It  liquefies  slowly. 
PATHOGENESIS. — It  is  without  effect,  even  in  very  large 

•doses. 

218 


DRAWINGS.  219 


Proteus  Vulgaris,  Hauser. 

ORIGIN. — It  is  very  widely  distributed  and  is  commonly 
present  in  the  putrefaction  of  animal  proteins  (p.  111).  It 
has  also  been  met  with  in  water,  in  meconium,  in  purulent 
abscesses,  and  in  the  blood  and  tissues  of  several  cases  of 
fatal  putrid  infection  of  the  intestines. 

FORM. — Rods,  of  varying1  length,  from  short  oval  forms 
to  those  which  are  2  to  6  times  as  long  as  wide.  It  is  usu- 
ally bent  and  grows  in  pairs;  may  also  form  twisted,  inter- 
woven threads.  Roundish  involution  forms  are  common. 

MOTILITY. — It  is  actively  motile.  Flagella  are  very 
numerous  (60-100),  and  are  arranged  all  over  the  surface. 

SPORULATION. — Not  observed,  though  cultures  are  re- 
sistant to  desiccation  and  retain  vitality  for  many  months. 

ANILIN  DYES. — It  stains  readily;  not  by  Gram's  method. 

GROWTH. — Very  rapid. 

Gelatin  plates.— Rapid  and  extensive  liquefaction  of  the  gelatin. 
The  colonies  are  yellowish  brown,  with  bristly  borders,  and  in  soft  gel- 
atin tend  to  spread  over  the  surface  and  assume  peculiar  figures.  De- 
tached portions  of  colonies  can  be  seen  to  move  about — "swarming 
islets."  A  disagreeable  odor  and  an  alkaline  reaction  is  present. 

Stab  culture.— Rapid  liquefaction  along  entire  line  of  inoculation, 
so  that  in  a  few  days  the  entire  contents  are  liquefied.  The  fluid  is  at 
first  diffusely  cloudy,  but  later  clears  up  and  a  flpcculent  sediment 
settles  on  the  bottom,  while  on  the  top  a  grayish  white  layer  is  formed. 

Streak  culture.—  On  agar,  it  forms  a  grayish,  slimy,  rapidly  spread- 
ing growth.  On  potato,  it  forms  a  dirty  colored,  sticky  covering. 

OXYGEN  REQUIREMENTS. — It  is  a  facultative  anaerobe. 

TEMPERATURE. — The  optimum  lies  between  20  and  24°. 
It  grows  excellently  in  the  incubator. 

BEHAVIOR  TO  GELATIN. — This  is  rapidly  liquefied. 

AEROGENESIS. — It  forms  hydrogen  sulphide. 

PATHOGENESIS. — Small  doses  have  no  effect.  The  injec- 
tion of  large  quantities  of  living,  or  filtered  cultures  pro- 
duces in  rabbits  and  guinea-pigs  toxic  effects,  and  death 
may  even  result.  It  is  therefore  toxicogenic.  At  times,  it 
may  even  take  on  a  pathogenic  character. 

This  and  several  related  species  are  included  in  the 
Bacterium  termo  of  the  older  writers. 

220 


DRAWINGS. 


221 


Bacterium  Zopfii,  Kurth. 

ORIGIN.— Isolated  from  the  intestines  of  chicken;  also 
from  water  and  feces. 

FORM. — Rods,  two  to  five  times  as  long  as  wide.  It 
forms  threads,  which  in  gelatin  are  often  peculiarly  bent 
or  twisted,  resembling  spirals.  In  old  cultures  coccus-like 
involution  forms  are  abundant. 

MOTILITY. — It  is  actively  motile. 

SPORULATION. — Spore-like  bodies  are  formed,  which 
are  said  to  resist  desiccation,  but  are  readily  destroyed  by 
heat,  and  are  readily  stained  by  anilin  dyes.  These  in 
reality  are  not  spores,  but  involution  forms. 

ANILIN  DYES. — It  is  stained  easily. 

G  RO  WTH.  — Rapid. 

Gelatin  plates. — The  colonies1  form  delicate  cloudy  patches  of 
radiating-  threads,  and  show,  under  the  microscope,  in  addition  to  the 
network  of  threads,  numerous  rounded  little  masses  or  bunches  of 
cells. 

Stab  culture. — Marked  growth  in  the  upper  part  of  the  tube,  but 
absent  from  the  lower  part.  It  shows  fine  radiating  lines  which,  at 
or  near  the  surface,  penetrate  deepest  into  the  surrounding  gelatin. 

Streak  culture. — On  agar,  it  forms  a  very  thin,  dry,  grayish  growth. 

OXYGEN  REQUIREMENTS. — It  is  an  obligative  aerobe. 

TEMPERATURE. — It  grows  best  at  ordinary  temperature. 
It  can  grow  at  37-40°,  but  tends  to  develop  involution 
forms  and  to  die  out. 

BEHAVIOR  TO  GELATIN. — No  liquefaction.     No  indol. 

PATHOGENESIS. — No  effect  has  been  observed  on  animals. 

This  organism  resembles  in  some  respects  certain 
species  of  the  Proteus  group  and,  indeed,  is  considered  by 
some  to  be  identical  with  the  P.  Zenkeri. 

1  When  surface  colonies,  as  those  above,  present  some  special 
characteristic  they  can  be  reprinted  on  cover-glasses.  To  make 
such  an  impression  or  "Klatsch"  preparation,  select  a  suitable 
spreading  colony,  with  the  aid  of  a  No.  3  objective,  then  raise  the  tube 
of  the  microscope  and  carefully  drop  a  clean  cover-glass  on  top  of 
the  colony.  Apply  gentle  pressure  with  a  pair  of  forceps,  then  grasp 
the  edge  of  the  cover-glass  and  carefully  remove;  allow  to  dry  in  the 
air;  fix  and  stain  in  the  usual  manner. 

In  making  the  reprint  only  the  growth  should  adhere  to  the 
cover-glass.  Considerable  gelatin,  solid  or  liquid,  on  the  cover-glass, 
is  undesirable  and  interferes. 

222 


DRAWINGS.  225 


Spirillum  Rubrum,  Esmarch. 

ORIGIN. — From  the  putrefied  cadaver  of  a  mouse. 

FORM.— Clear,  transparent,  thick  cells,  which  are 
usually  single,  appearing-  as  larg-e  bent  rods  or  comma 
bacilli  (vibrio).  It  may  form  spirals  of  three  or  four,  or 
even  forty  windings.  Involution  forms  are  common  in  old 
cultures. 

MOTILITY. — It  is  actively  motile.  Each  end  of  a  spiral 
has  one  wavy  nag^ellum. 

SPORULATION. — True  spores  have  not  been  observed. 

ANILIN  DYES. — These  stain  slowly  but  well,  especially 
if  the  dye  is  slightly  warmed. 

GROWTH. — This  is  extremely  slow. 

Gelatin  plates.— Owing  to  the  very  slow  development  of  colonies 
ordinary  plates  cannot  be  used.  In  roll-tubes,  colonies  develop  in  from 
seven  to  ten  days,  and  at  first  are  minute  and  grayish;  later  the  cen- 
ter of  each  colony  becomes  tinged  with  pink  and  eventually  becomes 
red.  The  edge  is  smooth  and  the  contents  are  finely  granular. 

Stab  culture.— This  is  the  most  characteristic.  Growth  takes 
place  along  the  entire  line  of  inoculation,  forming  a  row  of  colonies. 
The  growth  spreads  slightly  on  the  surface,  and  is  colored  a  light 
pink.  The  pigment  formation  is  most  marked  along  the  line  of  punc- 
ture— where  oxygen  is  absent.  It  passes  through  a  light  pink  to  a 
beautiful  dark  wine-red  color.  Ordinary  bacterial  pigments  are 
formed  only  in  the  presence  of  air,  and  are  secondary  products, 
whereas  this  pigment  is  formed  in  the  absence  of  air  and  is,  there- 
fore, a  primary  product. 

Streak  culture. — On  agar,  it  forms  moist,  thick,  non-spreading 
patches,  which,  when  old,  possess  a  light  pink  or  red  color,  especially 
near  the  center.  On  potato,  it  develops  slowly,  forming  minute 
•deep  red  colonies.  On  blood-serum,  the  growth  is  much  the  same  as 
on  agar. 

•Milk. — In  fluid  media,  milk,  bouillon,  etc.,  it  forms  long  spirals 
which  show  little  or  no  motion. 

OXYGEN  REQUIREMENTS. — It  is  a  facultative  anaerobe. 
TEMPERATURE. — It    grows    between   16°   and   40°.       The 
optimum  temperature  is  about  37°. 

BEHAVIOR  TO  GELATIN.— It  does  not  liquefy. 

PATHOGENESIS.— It  has  no  effect  on  animals. 

224 


DRAWINGS.  225 


Bacillus  Acidi  Lactici,   Hueppe. 

ORIGIN. — It  was  obtained  from  sour  milk. 

FORM. — Short,  thick  rods,  two  to  three  times  as  long 
as  wide;  these  are  usually  in  pairs;  rarely  in  chains  or 
threads. 

MOTILITY. — No  real  motion.  Marked  Brownian  move- 
ment. 

SPORULATION. — Round,  terminal  bodies  have  been  ob- 
served, but  these  are  not  spores. 

ANILIN  DYES. — It  stains  readily;  also  by  Gram's  method. 

GROWTH. — Abundant  and  rapid. 

Gelatin  plates. — The  deep  colonies  are  round  or  oval,  yellow,  sharp 
bordered,  finely  granular.  The  surface  colonies  spread,  forming-  thin 
plaques,  with  irregular,  wavy  borders.  The  outer  zone  of  the  colony 
is  at  first  almost  transparent  and  shows  marking's  resembling-  the  ven- 
ation of  leaves. 

Stab  culture. — Slig-ht  growth  along-  the  puncture,  but  on  the  surface 
it  is  considerable  and  spreads  rapidly  as  a  thin,  dry,  pearly-white  cov- 
ering. In  old  cultures  bundles  of  crystals  form  along-  the  line  of  inoc- 
ulation, especially  at  or  near  the  surface. 

Streak  culture. — On  a,gar,  it  forms  a  grayish  white,  moist,  spreading- 
growth,  which  offers  no  special  characteristics.  On  potato,  it  forms 
a  brownish  yellow,  slimy  covering. 

Milk.— -In  sterilized  milk  it  converts  the  lactose  or  milk-sug-ar,  in 
part,  into  lactic  and  carbonic  acids.  The  acid  reaction  thus  produced 
causes  a  precipitation  of  the  casein  or  curd.  This  chang-e  occurs  only 
in  the  presence  of  air.  Old  cultures  do  not  alter  milk — attenuation. 

OXYGEN  REQUIREMENTS. — It  is  a  facultative  anaerobe. 

TEMPERATURE. — It  grows  between  10°  and  45°.  The  op- 
timum is  at  about  30°. 

BEHAVIOR  TO  GELATIN. — The  latter  is  not  liquefied. 

AEROGENESIS. — Gas  is  produced  in  milk.  Carbon  diox- 
ide and  alcohol  are  formed. 

PATHOGENESIS. — No  effect.  0.75  per  cent,  lactic  acid 
stops  the  growth.  Production  of  lactic  acid  in  the  mouth 
(dental  caries);  abnormal  fermentations  in  the  stomach, 
in  the  intestines.  Lactic  acid  bacteria  favor  the  growth  of 
anaerobic  bacteria  (see  p.  100). 

The  cultural  characteristics  resemble  those  of  certain  colon 
bacilli  and  it  may,  therefore,  be  regarded  as  a  variety  of  the  B. 
aerogenes.  It  may  be  considered  as  a  common  cause  of  the  souring 
of  milk,  but  the  production  of  lactic  acid  is  common  to  a  larg-e  number 
of  bacteria,  and  hence,  such  organisms  will  also  coag-ulate  milk. 

226 


DRAWINGS.  227 


Bacillus  Butyricus,  Hueppe. 

ORIGIN. — Milk. 

FORM. — Long-,  narrow  rods,  with  rounded  ends,  fre- 
quently in  pairs,  may  form  threads. 

MOTILITY.  — It  is  actively  motile. 

SPORULATION. — At  about  30°  it  forms  bright,  oval,  median 
spores. 

ANILIN  DYES. — React  well. 

GROWTH.  — Rapid. 

Gelatin  plates. — The  deep  colonies  form  yellowish  masses,  whereas 
the  surface  ones  liquefy  rapidly  and  then  form  grayish-brown,  granu- 
lar patches  with  fibrillated  borders. 

Stab  culture. — Slow  liquefaction  along-  the  entire  line  of  inoculation. 
The  gelatin  becomes  colored  yellowish  and  on  the  surface  a  thin, 
folded,  grayish-white  scum  forms.  The  liquid  remains  cloudy  for  some 
time,  but  later  the  growth  settles  to  the  bottom. 

Streak  culture. — On  agar,  it  forms  a  thick,  grayish  or  yellow,  sticky 
covering-.  On  potato,  a  light  brown,  transparent  growth  results  which 
sometimes  becomes  folded. 

Milk. — Without  chang-e  in  the  amphoteric  reaction  the  casein 
gradually  coagnlates,  as  with  rennet.     Subsequently,   after  about  8 
days,  the  casein  is  redissolved  or  peptonized  with  formation  of  pepton, 
leucin,  tyrosin,  ammonia  and  bitter  products.     From  hydrated  milk 
sugar  and  lactates  it  forms  butyric  acid. 

OXYGEN  REQUIREMENTS. — It  is  aerobic. 
TEMPERATURE. — It  can  grow  at  the  ordinary  temperature, 
but  its  optimum  is  at  35  to  40°. 

BEHAVIOR  TO  GELATIN. — It  liquefies  gelatin. 
AEROGENESIS. — Butyric  acid  is  formed. 
PATHOGENESIS.  — It  has  no  effect  on  animals. 

A  large  number  of  aerobic  and  anaerobic  bacteria  give 
rise  to  butyric  acid  fermentation  (see  p.  103).  The  vibrion 

butyrique  of  Pasteur  was  the  first  anaerobe  discovered  (1861). 

228 


DRAWINGS.  229 


Bacillus  Cyanogenus,  Fuchs  (1841). 

BACILLUS  OF  BLUE  MILK. 

ORIGIN. — It  was  obtained  from  blue  milk. 

FORM. — Small,  rather  narrow  rods,  with  slig-htly 
rounded  ends,  2  to  3  times  as  long-  as  wide.  It  frequently 
grows  in  pairs,  very  rarely  in  threads. 

MOTILITY. — Very  actively  motile.  Bunch  of  whips  at 
one  end. 

SPORULATION.  — The  small  terminal  spore-like  bodies 
observed  by  some  are  probably  involution  forms.  True 
spores  have  been  observed  to  form  on  althea  or  quince  jelly. 

ANILIN  DYES. — It  stains  easily. 

GROWTH.  — Rapid. 

Gelatin  plates. — The  deep  colonies  are  round  with  sharp,  smooth 
border,  and  yellowish  granular  contents.  The  surface  colonies  are 
moist,  elevated,  convex  masses,  which  are  round,  finely  granular  and 
dark  colored.  At  times  the  colonies  may  be  thin,  spreading"  and  with 
an  irregular  border. 

Stab  culture. — Little  or  no  growth  in  the  lower  part  of  the  punc- 
ture. It  spreads  over  the  surface  as  a  thick,  moist,  dark-gray  cover- 
ing-. A  dark  steel-blue  color  diffuses  downward  into  the  gelatin.  The 
shade  of  color  varies  with  the  reaction  of  the  medium.  In  neutral 
or  acid  media  it  is  quite  blue,  whereas  in  very  alkaline  media  it  is 
dark,  or  even  black.  The  culture  when  old  becomes  dark  colored. 

Streak  culture.— On  agar,  it  forms  a  dirty  gray,  thick,  moist  cover- 
ing-, and  the  medium  becomes  dark  colored.  On  potato,  it  likewise 
forms  a  thick,  raised,  slimy  growth,  which  rapidly  spreads  and  be- 
comes colored,  On  blood-serum,  no  color  is  formed. 

Milk.— In  sterilized  milk  it  produces  no  acid  or  coagulation,  but 
the  liquid  becomes  colored  a  slate-gray  which  with  acids  turns  blue. 
In  unsterilized  milk,  that  is  in  presence  of  lactic  acid  bacteria,  the 
color  is  sky-blue.  The  color  is  developed  from  casein,  not  from 
lactose.  In  bouillon  or  milk  containing  2  per  cent,  of  glucose  it  forms  a 
splendid  blue  color  and  lactic  acid.  It  does  not  convert  lactose  into 
an  acid. 

OXYGEN  REQUIREMENTS. — Aerobic. 

TEMPERATURE. — It  grows  best  at  the  ordinary  tempera- 
ture; to  less  extent  in  the  incubator.  The  pigment  is  most 
marked  when  growth  occurs  at  low  temperatures,  15-18°. 

BEHAVIOR  TO  GELATIN. — This  is  not  liquefied. 

PATHOGENESIS. — It  has  no  effect  on  animals. 

The  production  of  a  blue  color  in  milk  is  frequently  observed, 
and,  as  in  the  case  of  red  milk,  this  is  due  to  the  development  of 
certain  species  of  bacteria. 

230 


DRAWINGS.  231 


CHAPTER  IX. 


BOUILLON,  AGAR,    MILK  AND   MODIFIED   MEDIA— THE  INCUBA- 
TOR   AND  ACCESSORIES. 


The  work,  thus  far,  has  enabled  the  student  to  acquire 
familiarity  with  the  fundamental  methods  of  bacteriology. 
The  various  non-pathogenic  bacteria  have  been  isolated  by 
means  of  gelatin  plates;  their  characteristics  of  growth  in 
the  colony,  in  stab  culture,  and  on  potato,  have  been  stu- 
died. A  special  bread  medium  has  been  employed  in  culti- 
vating the  several  important  moulds.  The  gelatin,  potato 
and  bread  media  are,  however,  by  no  means  the  only  ones 
employed  in  growing  micro-organisms. 

While  the  nutrient  gelatin  is  invaluable  for  the  isola- 
tion of  certain  bacteria,  especially  those  of  the  saprophytic 
group,  it  is  not  so  generally  useful  in  connection  with  the 
pathogenic  bacteria.  Many  of  these  grow  only,  at  or  near 
the  temperature  of  the  body  (37.5°),  and,  inasmuch  as  the 
ordinary  gelatin  melts  at  about  25°,  it  cannot  obviously  be 
employed  to  obtain  colonies,  or  stab  cultures,  of  such  or- 
ganisms. 

It  is,  therefore,  desirable  to  have  a  substance  which, 
to  a  certain  extent,  can  take  the  place  of  gelatin;  one  which 
will  make  a  medium  such  that  it  will  not  melt,  even  at  the 
temperature  of  the  body.  The  sea-weed  agar-agar,  which 
is  gathered  off  the  coast  of  Asia,  and  about  the  islands  of 
the  Pacific,  answers  this  requirement.  A  more  than  two 
per  cent,  solution  of  agar  is  not  made,  and  is  not  desirable. 
The  prepared  agar  medium  melts  at  the  temperature  of 
boiling  water  (100°),  and  becomes  solid  again  when  the 
temperature  is  reduced  to  about  40°.  This  medium,  there- 


BOUILLON,  AGAR  AND  MILK.  233 

fore,  is  extremely  useful  in  the  study  of  pathogenic  organ- 
isms. Unlike  gelatin,  it  is  not  liquefied  by  bacteria  except 
perhaps  in  a  very  few  rare  cases,  and  hence,  it  can  not 
supplant  gelatin  in  the  study  and  differentiation  of  bac~ 
teria. 

In  addition  to  the  solid  media,  it  is  desirable  to  have  a 
liquid  medium.  The  beef-tea  or  bouillon,  prepared  accord- 
ing to  the  following  directions,  is  extremely  useful  for  this 
purpose.  Indeed,  before  the  introduction  of  solid  media,  it 
was  the  chief  medium  employed.  The  growths  in  beef -tea 
are  frequently  very  characteristic,  and  materially  assist  in 
the  recognition  of  various  bacteria. 

The  meat  extract  employed  in  the  preparation  of  gela- 
tin, agar  and  bouillon,  is  usually  made  out  of  beef.  The 
Pasteur  school,  almost  invariably,  use  veal  for  the  prepara- 
tion of  these  media.  The  reason  for  this  lies  in  the  greater 
amount  of  extractive  substances  contained  in  veal.  For 
^special  purposes,  bouillon  etc.,  is  made  out  of  chicken, 
pork,  or  other  flesh.  The  commercial  meat  extracts  can 
be  employed  for  making  bouillon  and  the  other  media 
(see  p.  243). 

Milk  constitutes  another  important  liquid  medium.  On 
account  of  the  presence  of  lactose  and  casein,  milk  is  espe- 
cially valuable  in  differentiating  certain  bacteria.  Some 
bacteria  will  decompose  the  milk-sugar  and  cause  the  evo- 
lution of  gas-bubbles,  whereas  others  will  not  do  so.  Again, 
some  will  cause  coagulation,  as  is  seen  in  sour  milk,  while 
other  bacteria  produce  no  appreciable  change. 

Consequently,  before  taking  up  the  study  of  pathogenic 
bacteria,  the  student  will  prepare,  in  addition  to  the  gela- 
tin, and  potato  media,  already  on  hand,  the  necessary  bouil- 
lon, agar,  and  milk  media  (see  p.  243). 


234  BACTERIOLOGY. 

Bouillon. 

The  meat  extract  is  prepared  according*  to  the  directions 
given  under  the  preparation  of  gelatin  (p.  153).  To  1 
liter  of  the  meat  extract,  10  g.  of  dry  pepton  (Witte's)  and 
5  g.  of  common  salt  are  added.  The  liquid  is  then  warmed 
at  55-60°,  with  constant  stirring,  till  the  pepton  dissolves. 
It  is  then  rendered  alkaline  according  to  the  directions 
given  on  p.  154.  The  portions  of  10  c.c.  of  the  solution  in 
test-tubes  should  receive  0.4,  0.6,  0.8  and  1.0  c.c.  of  the  -fs 
NaOH,  respectively.  The  neutral  point  is  usually  reached 
by  the  addition  of  about  0.5  c.c.  &  NaOH  per  10  c.c.  The 
remaining  liquid  is  measured  and  amount  of  N  NaOH  nec- 
essary to  neutralize  the  entire  amount  is  then  calculated. 
This  calculated  amount,  plus  10  c.c.  of  N  NaOH  per  liter,  in 
order  to  impart  a  slight  alkalinity,  is  then  added.  The 
liquid  is  then  placed  in  the  enamelled  jar  (Fig.  23,  p.  153), 
and  immersed  in  a  boiling  water-bath,  or  boiled  directly 
over  the  flame  for  ^  an  hour.  Inasmuch  as  there  will  be 
a  considerable  loss  of  water  by  evaporation,  it  is  advisable 
to  mark  the  level  of  the  liquid,  before  boiling.  At  the 
close  of  the  operation  sufficient  distilled  water  should  be 
added  to  bring  the  liquid  back  to  the  original  volume.  A 
better  procedure  is  to  weigh  the  liquid,  before  and  after 
boiling,  and  for  the  number  of  grams  lost  in  weight  to  add 
a  corresponding  number  of  c.c.  of  distilled  water. 

The  liquid  is  then  filtered  through  a  wet  filter.  The 
filtrate  is  usually  perfectly  clear  and  transparent,  but  it. 
not  infrequently  happens,  when  it  is  placed  in  tubes  and 
sterilized,  that  a  fine  amorphous  precipitate  will  form. 
Usually  this  is  unimportant,  but,  at  times,  it  is  desirable  to 
have  a  bouillon  which  will  remain  perfectly  clear,  free  from 
the  slightest  deposit.  To  obtain  this  result,  the  filtered 
bouillon  is  returned  to  the  clean,  enamelled  jar  and  sub- 
jected to  more  heat.  It  can  be  placed  in  the  autoclave  and 


BOUILLON,  AGAR  AND  MILK.  235- 

heated  for  i  an  hour  at  110°,  or,  what  is  even  more  prefer- 
able, it  is  concentrated  over  a  flame  to  less  than  |  its 
original  volume.  The  vessel  is  finally  weighed,  and  the 
distilled  water  necessary  to  bring  back  the  bouillon  to  the 
original  weight  is  added.  The  bouillon  is  again  filtered 
and  the  filtrate  is  then  filled  into  tubes.  The  small  test- 
tubes  (12  x  150  mm.)  are,  as  a  rule,  desirable  for  thi& 
purpose.  These  are  plugged  and  sterilized  in  the  usual 
manner. 

The  bouillon  is  then  sterilized  according  to  the  direc- 
tions given  under  gelatin  (p.  162);  that  is  to  say,  it  is 
exposed  to  steam-heat  for  15  minutes  on  each  of  three 
consecutive  days. 

The  bouillon  prepared  as  above  should  be  (1)  perfectly 
clear;  (2)  should  be  slightly  alkaline  in  reaction;  and,  (3) 
should  not  cloud  on  heating. 

Agar-Agar. 

One  liter  of  bouillon  is  prepared  as  above,  except  that 
the  second  heating  is  omitted.  20  g.  of  agar  (2  per  cent.) 
are  cut  up  into  as  small  pieces  as  possible,  and  these  are 
then  added  to  the  bouillon  which  has  been  returned  to  the 
clean  jar.  The  jar  and  contents  are  weighed,  and  then 
placed  over  a  free  flame  to  boil.  The  heat  should  be  just 
sufficient  to  gently  boil  the  liquid,  otherwise  the  liquid  is 
liable  to  froth  and  run  over.  The  liquid  is  boiled  for  i  -  f  of 
an  hour,  or  until  the  agar  has  completely  dissolved.  This 
point  can  be  readily  ascertained  by  drawing  up  the  liquid 
into  a  clean  glass  tube  or  pipette.  No  solid,  translucent 
lumps  should  be  visible.  The  jar  and  contents  are  now 
weighed,  and  distilled  water  is  added  to  replace  that  lost 
by  evaporation. 

The  jar  is  then  placed  in  the  steam  sterilizer;  or,  better, 
immersed  in  a  water-bath  at  a  constant  temperature  of 


236  BACTERIOLOGY. 

about  50°  1.  In  an  hour  or  two,  the  finely  divided,  insoluble 
matter  gathers  into  masses  and  settles  to  the  bottom, 
leaving-  above  a  clear,  transparent  liquid. 

The  filtration  of  2  per  cent,  agar  through  paper  is 
usually  a  very  slow,  tedious  and  unsatisfactory  operation. 
Consequently,  it  is  usually  filtered  either  through  a  layer 
of  cotton,  or  else,  after  sedimentation,  the  agar  is  allowed 
to  solidify  and  then  the  clear  portion  is  cut  off  while  the 
bottom  layer,  containing  the  impurities,  is  discarded.  By 
employing  the  following  method,  a  liter  of  agar  can  be 
filtered  perfectly  clear,  or  as  clear  as  is  necessary  for  all 
purposes,  in  from  3  to  5  minutes. 

The  filtering  arrangement  is  shown  in  Fig.  35.  The  re- 
ceiving flask  (1^  1.  capacity)  is  connected  with  a  Chapman 
aspirator.  The  neck  receives  the  rubber  stopper,  which  is 
slipped  over  the  end  of  a  funnel.  A  Witte's  porcelain,  per- 
forated plate  (10  cm.  in  diameter)  is  placed  in  the  funnel, 
and  steadied  in  position  by  the  glass  rod  which  passes 
through  the  center.  A  disc  of  muslin  having  the  same 
diameter  as  the  plate  (or  a  trifle  less)  is  placed  on  the  lat- 
ter. Then  a  circle  of  absorbent  cotton,  about  12  cm.  in 
diameter,  is  placed  on  top,  and  finally  another  layer  of 
muslin.  A  second  Witte's  plate  is  now  placed  on  top,  as  a 
weight,  but  it  remains  there  only  during  the  preliminary 
warming  up  of  the  funnel.  About  half  a  liter  of  boiling 
water  is  then  poured  into  the  funnel.  The  pump  is  set  into 
action,  and  the  hot  water  rapidly  passes  through  the  filter. 
It  is  returned  to  the  funnel,  several  times  in  succession, 
thus  warming  up  the  funnel,  filter  and  flask.  During  this 
operation,  care  should  be  taken  to  see  that  the  cotton 
closes  tight  over  the  edge  of  the  porcelain,  and  that  no 
openings  exist.  While  the  pump  is  still  active,  the  upper 
plate  is  removed.  The  suction  of  the  pump  draws  down 
the  filter,  and  hence  the  weight  is  no  longer  necessary. 

1It  is  advisable  to  place  the  jar  in  a  water-bath  which  is  pro- 
vided with  a  thermo-regulator  (see  Fig.  75). 


BOUILLON,  AGAR  AND   MILK. 


237 


While  the  filter  is  still  hot,  and  the  pump  active,  the 
well  sedimented  agar  (p.  236)  is  slowly  poured  on  the  cen- 
ter of  the  filter.  The  liquid  passes  through  as  rapidly  as  it 
strikes  the  surface.  The  impurities  are  brought  last  upon 
the  filter.  If  the  filtrate  is  not  perfectly  clear,  it  can  be 
passed  through  the  filter  a  second  or  a  third  time.  The 
operation  of  filtration  requires  but  a  few  minutes.  The 
resulting  filtrate,  when 
filled  into  tubes  and 
sterilized,  is  usually 
wholly  free  from  a  de- 
posit, and  is  perfectly 
transparent.  At  most, 
a  few  floccules  will 
separate  during  the 
subsequent  steriliza- 
tion. 

In  order  to  facili- 
tate the  filtration 
through  paper  many 
workers  prefer  to 
make  a  1  or  1^  per 
cent,  solution  of  agar. 
While  it  is  true  that 
such  solutions  filter 
more  readily,  yet  this 

advantage  is  more  than  offset  by  the  softer  consistency 
of  the  agar.  The  two  per  cent,  agar  prepared  as  above  is 
hard,  and  though  it  is  not  filtered  through  paper,  it  is,, 
nevertheless,  perfectly  clear. 

The  above  method  of  making  agar  would  seem  to  be 
long  and  tedious,  but  in  reality,  it  is  no  more  difficult  than 
the  preparation  of  gelatin.  While  the  pump  is  very  useful, 
in  the  above  filtration,  it  is  by  no  means  necessary. 

In  the  method  as  described,  the  agar  is  not  added  to- 
the  albuminous  meat  extract,  but  to  the  ready-made  bouil- 


FIG.  35.    Apparatus  for  filtering  agar  (F.  G.  N.). 


238  BACTERIOLOGY. 

Ion.  If  it  were  added  to  the  former,  the  agar  would  dis- 
solve much  more  slowly  than  in  clear  bouillon,  and,  more- 
over, the  coagulated  albumin  would  materially  interfere 
with  the  subsequent  filtration.  As  it  is,  only  the  impurities 
in  the  agar  are  to  be  filtered  off.  The  agar  is  neutral  in 
reaction,  and  hence,  it  is  added  to  the  already  alkalized 
bouillon. 

The  filtered  agar  is  filled,  by  means  of  a  small  funnel, 
into  plugged,  sterilized  test-tubes,  and  these  are  then  ster- 
ilized by  steaming  for  30  minutes,  on  each  of  three  succes- 
sive days.  As  many  tubes  as  are  necessary  for  immediate 
use  are  inclined  by  placing  on  a  thick  glass  rod,  or  narrow 
strip  of  wood,  or  on  the  gas  tubing.  The  small  test-tubes 
{12  X  150  mm.)  are  preferable  for  agar.  The  remaining 
tubes  are  kept  in  the  upright  position,  and  are  inclined 
whenever  it  is  necessary.  Agar  may  be  used,  either,  in  the 
inclined  position,  for  streak  cultures,  or  in  the  upright  posi- 
tion, for  stab  cultures.  It  may  be  mentioned  incidentally 
that  inclined  gelatin  may  often  be  employed  to  advantage. 

The  agar  solidifies  in  this  inclined  position.  A  large 
surface  can  thus  be  obtained,  which  can  be  used  for  making 
a  streak  culture,  or  even  for  the  isolation  of  colonies.  The 
streak  culture  is  made,  as  in  the  case  of  those  on  potato 
{p.  186),  by  drawing  the  inoculated  wire  along  the  middle 
of  the  inclined  surface.  In  describing  the  use  of  the  potato 
tube  (p.  186)  it  was  shown  that  these  could  be  used  for  ob- 
taining isolated  colonies.  The  same  method  can  be  applied 
to  the  inclined  agar.  It  enables  one  to  obtain  isolated 
colonies  in  less  than  twenty-four  hours,  and  that,  without 
the  use  of  any '  special  plating  apparatus.  A  very  small 
amount  of  the  material  is  taken  up,  on  a  platinum  wire, 
and  spread  over  the  entire  inclined  surface  of  an  agar  tube. 
The  same  wire,  without  sterilization,  is  then  rubbed  over  the 
inclined  surface  of  a  second  tube,  and  then  in  like  manner 
over  a  third  and  fourth.  The  tubes  are  then  placed  in  an 
incubator  to  develop. 


BOUILLON,  AGAR  AND  MILK.  239 

If  the  material  used  is  a  solid  growth,  it  is  advisable  to 
transfer  a  minute  amount  of  this  to  a  tube  of  bouillon 
(10  c.c.).  With  a  sterilized  wire,  a  loopful  of  this  suspension- 
is  transferred  to  a  second  tube  of  bouillon.  The  wire  is  again 
sterilized,  and,  a  loopful  of  this  second  dilution  is  trans- 
ferred to  a  tube  of  freshly  inclined  agar  and  carefully  spread 
all  over  the  surface.  The  same, wire,  not  sterilized,  is  then 
rubbed  over  the  surface  of  a  second  tube  of  inclined  agar. 
The  inoculated  agar  tubes  should  be  placed  in  a  horizontal 
position  in  the  incubator  for  18  to  24  hours.  Perfectly  iso- 
lated colonies  will  thus  be  obtained. 

The  method  of  isolating  colonies  by  means  of  agar  plates 
will  be  described  in  the  next  chapter.  Esmarch  roll-tubes 
(p.  181),  can  be  made  with  agar  in  the  same  manner  as  with 
gelatin.  The  tubes  should  be  rolled  in  ordinary  tap-water 
and  not  in  ice-water.  They  should  then  be  set  aside  in  a 
cool  place,  in  a  horizontal  position,  in  order  to  allow  the 
agar  to  adhere  firmly  to  the  glass. 


Milk. 


This  should  be  obtained  as  fresh  as  possible,  before  the 
reaction  has  become  acid.  It  is  then  filled  into  sterile 
tubes  and  sterilized  by  exposure  to  steam  for  30  minutes  on 
each  of  three  successive  days.  It  can  be  sterilized  in  the 
autoclave  at  110°-115°  for  10-15  minutes.  Too  much  heat 
should  be  avoided,  since  this  may  cause  an  alteration  of  the 
milk.  It  should  be  remembered,  however,  that  milk  is  dif- 
ficult to  sterilize  because  of  the  presence  of  resistant  organ- 
isms, and,  above  all,  because  of  the  protection  afforded  by 
the  fat.  Hence,  it  is  advisable  to  place  the  milk  tubes  in 
the  incubator  at  37°  for  1-2  days  in  order  to  make  sure  of 
the  sterility  of  the  medium, 

In  the  method  as  described,  whole  milk  is  employed, 
and  hence,  on  standing,  a  thick  layer  of  cream  rises  to  the 
surface.  This  can  be  avoided  by  employing  centrifugated 


240  BACTERIOLOGY. 

milk;  or,  if  that  cannot  be  obtained,  the  milk  should  be 
skimmed.  In  order  to  avoid  fermentative  changes,  the 
milk  should  be  steamed  for  at  least  15  minutes.  It  can 
then  be  set  aside  in  a  cool  place  for  the  cream  to  rise.  By 
means  of  a  100  c.c.  bulk  pipette  the  thin  milk  can  be  drawn 
off  from  the  bottom  and  filled  into  tubes. 

Milk  is  used  in  order  to  ascertain  whether  an  organism 
can  coagulate  casein,  and  whether  it  can  decompose  the 
lactose  thus  giving  rise  to  gas  bubbles,  or  to  acid  pro- 
ducts. The  first  two  changes  can  be  observed  in  the  ordi- 
nary sterile  milk.  To  detect  the  presence  of  acid  products  it 
is  necessary  to  color  the  milk  with  blue  litmus.  This  can 
be  done  by  adding  a  concentrated  litmus  solution  to  the 
milk  before  sterilization;  or,  by  adding  the  sterile  litmus  so- 
lution to  the  sterilized  milk,  by  means  of  a  drawn  out 
pipette  (Fig.  61). 

Modified  Media. 


The  ordinary  nutrient  agar,  bouillon,  or  gelatin,  pre- 
pared according  to  the  directions  heretofore  given,  may  be 
greatly  improved  as  nutrient  media  for  certain  organisms 
by  the  addition  of  glycerin,  glucose,  lactose,  litmus,  etc. 

Peptonless  agar. — This  is  ordinary  agar  minus  the  pep- 
ton.  It  is  especially  useful  when  it  is  desired  to  obtain 
spores,  as  in  the  case  of  anthrax. 

Glycerin  media. — Usually  5  percent,  of  glycerin  is  added 
to  bouillon,  or  to  agar.  In  the  case  of  Roux  potato  tubes 
(Fig.  34  &,  p.  184),  the  lower  bulb  may  be  filled  with  a  5  per 
cent,  aqueous  solution  of  glycerin.  The  addition  of  glycerin 
renders  these  media  especially  useful  for  the  cultivation  of 
the  tubercle  bacillus.  The  streptococci,  and  the  bacteria  of 
diphtheria,  glanders  and  pneumonia  likewise  thrive  exceed- 


MODIFIED  MEDIA.  241 

ingly  well  on  glycerinated  media.  The  manner  in  which 
glycerin  favors  the  growth  of  the  tubercle  bacillus  is  not 
known,  but  inasmuch  as  this  organism  stores  up  within  its 
cell  a  large  amount  of  fat,  it  would  seem  as  if  the  glycerin 
was  directly  utilized  for  the  making'  of  this  compound. 

Glucose  media. — The  addition  of  2  per  cent,  of  glucose  to 
bouillon,  gelatin,  or  agar  greatly  improves  these  as  media 
for  the  cultivation  of  anaerobic  bacteria.  Glucose  is  a  pow- 
erful reducing1  substance,  taking"  up  oxygen  from  its  neigh- 
borhood. In  this  way,  it  is  supposed  to  favor  the  growth  of 
these  bacteria.  Again,  certain  bacteria  readily  ferment 
glucose,  giving-  rise  to  acid,  and  to  gaseous  products.  Hence, 
the  addition  of  glucose  is  of  great  value  in  differentiating 
species.  Sometimes  litmus  is  added  to  glucose  media  in  or- 
der to  indicate  the  reaction  produced  by  the  organism.  Lit- 
mus glucose  gelatin  is  especially  useful  for  cultivating 
anaerobic  bacteria,  with  or  without  a  special  apparatus. 
Moreover,  the  vitality  of  these  organisms  are  greatly  pro- 
longed in  this  medium. 

Lactose  media. — Instead  of  glucose,  2  per  cent,  of  milk- 
sugar  may  be  added  to  the  several  media.  Usually,  the  lac- 
tose media  are  colored  at  the  same  time  with  litmus.  Cer- 
tain bacteria,  like  the  typhoid  bacillus,  cannot  ferment  lac- 
tose, and  hence  the  reaction  remains  alkaline  and  no  gas 
bubbles  are  given  off.  On  the  other  hand,  some  bacteria 
like  the  colon  bacillus,  do  ferment  lactose,  in  which  case  the 
litmus  turns  red  and  gas  bubbles  appear.  Lactose  media 
are  therefore  valuable  as  a  means  of  differentiating  organ- 
isms. 

Litmus  media. — The  commercial  litmus  usually  contains 
a  large  admixture  of  red  pigment  which  interferes,  more  or 
less,  with  the  delicacy  of  the  reaction.  It  is  advisable, 
therefore,  to  purify  the  litmus.  This  can  be  done  according 

16 


242  BACTERIOLOGY. 

to  the  method  given  in  Chapter  XV.  Litmus  is  usually  ad- 
ded to  the  glucose,  or  lactose  media,  or  to  milk  in  order  to 
ascertain  whether  a  given  organism  produces  acid  products. 
The  medium  should  be  colored  decidedly  blue.  A  very  con- 
centrated aqueous  solution  of  litmus  should  be  employed  in 
order  not  to  dilute  the  medium  too  much.  Excessive  steril- 
ization of  sugar  media,  which  contain  litmus,  results  in  the 
decoloration  of  the  latter.  The  color  may,  in  part,  return 
on  standing*.  It  is  advisable,  therefore,  not  to  steam  for 
more  than  15  minutes  at  a  time;  or  better  still,  to  sterilize 
the  litmus  solution  by  itself  and  then  add  this,  by  means  of 
a  sterile  pipette  (Fig.  61),  to  the  sterile  nutrient  medium. 

Serum  media. — The  preparation  of  blood-serum,  and  of 
serous  exudates  will  be  described  later.  In  this  connection 
it  will  be  sufficient  to  state  that  the  addition  of  blood-serum 
from  animals,  or  of  highly  albuminous  exudates  obtained 
from  man,  makes  an  agar  or  bouillon  specially  valuable  for 
the  cultivation  of  certain  bacteria. 

Thus  Lfiffler's  blood-serum  which  is  an  excellent  medium 
for  the  diphtheria  bacillus,  consists  of  3  parts  of  blood- 
serum  and  1  part  of  a  1  per  cent,  glucose  bouillon. 

The  addition  of  1  part  of  ascitic  fluid  to  2  parts  of  agar 
gives  a  medium  well  adapted  for  cultivating  the  gonococcus. 
The  fluid,  must  of  course,  be  collected  under  sterile  condi- 
tions, otherwise  it  should  be  filtered  through  porcelain 
(Fig.  66).  Such  a  mixture  can  be  used  for  plating  purposes, 
in  which  case,  however,  the  agar  should  be  first  melted  and 
allowed  to  cool  to  about  50°.  The  ascitic  fluid,  or  blood- 
serum,  previously  warmed  to  this  temperature,  should  then 
be  added  to  the  liquid  agar.  The  mixture  can  now  be  inocu- 
lated, dilutions  made  and  Petri  dishes  poured  as  in  the  case 
of  ordinary  agar  cultures. 

Laboratory  work.— One  half  of  the  class  will  prepare  agar  and 
bouillon  according-  to  the  methods  just  given.  One  liter  of  bouillon  is 
prepared  first,  and  from  this  the  agar  and  the  modified  media  are  pre- 


THE  INCUBATOR  AND  ACCESSORIES.  243 

pared  in  the  quantities  indicated  below.  The  other  half  will  prepare 
the  same  media  using-,  however,  commercial  meat  extract  instead  of 
fresh  beef.  The  method  is  as  follows: 

To  1000  c.c.  of  water  add  10  g.  of  dry  pepton  (Witte's),  5  g.  of  com^ 
mon  salt,  and  2.5  g.  of  Liebig's  meat  extract,1  warm  till  solution  re. 
suits,  then  alkalize  according-  to  the  directions  given  (p.  234).  Boil  for 
half  an  hour  and  replace  the  water  lost  by  evaporation,  then  filter. 

Take  100  c.c.  of  the  bouillon  and  fill  into  the  small  sterilized  test- 
tubes,  using1  a  small  funnel,  to  a  depth  of  li  inches  =  ordinary  bouil- 
lon. 

To  100  c.c.  of  the  bouillon,  add  2  per  cent,  of  glucose  and  warm  till 
dissolved.  Then  fill  this  into  test-tubes  =  glucose  bouillon. 

To  the  remainder  (800  c.c.),  add  2  per  cent,  of  finely  cut  ag-ar  and 
boil  over  a  flame  till  it  dissolves.  Then  sediment  and  filter  according 
to  the  directions  already  given  (p.  236). 

To  100  c.c.  of  the  filtered  agar,  add  5  per  cent,  of  pure  glycerin 
and  mix  thoroughly.  Test  the  reaction  in  order  to  make  sure  that  it 
is  alkaline.  Finally,  fill  into  small,  sterilized  test-tubes  =  glycerin 
agar. 

To  100  c.c.  of  the  filtered  agar,  add  2  per  cent,  of  glucose  and  warm 
till  it  dissolves.  Then  fill  into  tubes  to  a  depth  of  about  2  inches  = 
glucose  agar. 

Transfer  the  remainder  of  the  agar  to  sterilized  test-tubes  =  or- 
dinary agar.  The  several  bouillon  and  agar  media  are  then  fraction- 
ally sterilized  (p.  162). 


The    Incubator. 

In  cultivating  pathogenic  organisms  it  is  advisable  to 
grow  these,  whenever  possible,  at  the  temperature  of  the 
body.  In  this  way,  they  are  supplied  with  one  of  the  con- 
ditions under  which  they  live  in  nature.  A  few  organisms, 
notably  the  tubercle  bacillus  and  the  gonococcus,  will  show 
no  growth  at  the  ordinary  temperature.  In  such  instances, 
it  is  absolutely  necessary  to  use  a  higher  temperature  in  or- 

1  Most  any  of  the  commercial  meat  extracts  will  answer  equally 
well.  A  nutrient  gelatin  can  be  prepared  with  such  extracts  in  the 
same  way  as  agar. 


244  BACTERIOLOGY. 

der  to  obtain  results.  The  apparatus  employed  for  this 
purpose  is  known  as  an  incubator  or  thermostat.  It  is  made 
of  heavy  copper  and  has  double  walls.  The  space  between 
these,  which  should  be  as  large  as  possible,  is  filled  with 
distilled  water.  In  form  the  thermostat  may  be  oblong, 
square  or  oval.  Fig.  36  shows  an  excellent  thermostat  of 
oval  form. 


FIG.  36.    The  incubator  or  thermostat. 

It  is  desirable  that  the  incubator  should  have  as  much 
available  space  as  possible  for  dishes,  flasks,  tubes,  etc. 
The  apparatus  as  ordinarily  made  is  quite  expensive.  Any 
one  with  a  little  ingenuity  can  plan  out  a  double -walled  box 
and  have  an  incubator  made  by  a  tinsmith  which  will  answer 
all  ordinary  purposes.  It  should  have  a  double  door  or  lid, 
and  it  is  advisable,  though  not  necessary,  that  the  side  and 
top  should  be  covered  with  asbestos  or  felt.  The  top  should 
be  provided  with  an  opening  which  communicates  with  the 
water  compartment  and  serves  to  hold  a  thermo-regulator. 


THE  INCUBATOR  AND  ACCESSORIES.  245 

Another  opening  serves  to  communicate  with  the  interior  of 
the  incubator  and  receives  a  thermometer. 

A  large,  well-equipped  laboratory  should  have,  in  addi^ 
tion  to  a  number  of  the  ordinary  thermostats,  at  least  one 
incubating  room.  That  is  to  say,  a  room  which  is  maintained 
at  a  constant  body  temperature.  This  can  be  heated  by 
means  of  a  small  cylindrical  stove,  which  is  heated  by  a  gas 
burner  and,  from  which  hot  air  pipes  radiate  to  different 
parts  of  the  room,  A  more  constant  temperature  can  be 
maintained  by  passing  hot-water  pipes  around  the  walls  of 
the  room.  The  water  is  heated  on  the  outside  of  the  room 
by  means  of  a  suitable  burner. 

Thermo-regulators. — In  order  that  a  thermostat  shall 
maintain  a  constant  temperature,  it  is  necessary  that  it 
shall  be  provided  with  a  good  thermoregulator.  This  instru- 
ment automatically  regulates  the  supply  of  gas.  The  bulb 
of  the  instrument,  containing  alcohol  or  mercury,  is  im- 
mersed in  the  water  between  the  walls  of  the  incubator. 
When  the  temperature  rises,  the  alcohol  or  mercury  expand 
and  shut  off  the  supply  of  gas.  In  order  to  prevent  the 
gas  from  being  cut  off  completely,  a  minute  opening  is 
made  in  the  delivery  tube,  thus  permitting  the  passage  of 
a  small  amount  of  gas,  which  is  sufficient  to  maintain  a 
small  light. 

The  simplest  mercury  thermo-regulator  is  that  of 
Reichert.  As  ordinarily  made,  it  is  the  exception  to  find  a 
Reichert  regulator  that  will  work  properly.  The  minimum 
opening  is  invariably  too  large,  and  will,  therefore,  allow 
the  passage  of  more  gas  than  is  necessary  to  maintain  the 
the  desired  temperature.  In  order  to  obviate  this  difficulty, 
a  number  of  modifications  of  the  Reichert  instrument  have 
been  made,  and  some  of  these  answer  the  purpose  very 
well.  Unfortunately,  the  diameter  of  the  delivery  tube,  as 
usually  made,  is  narrow  and  hence  such  a  regulator  can  be 
used  only  for  low  temperatures. 


246 


BACTERIOLOGY. 


The  author's  thermo-regulator ',  shown  in  Fig1.  37,  is 
intended  to  overcome  the  difficulties  mentioned.  Although, 
apparently,  more  complicated  than  the  ordinary  regulator, 
it  nevertheless  is  easily  adjusted  and  is  extremely  sensitive. 
The  gas  tubing-  is  attached  to  the  strong,  wide  inflow  and 

outflow  tubes  (a!  and  a"}  and  is 
thus  independent  of  the  regulat- 
ing parts.  The  upper  portion 
consists  of  the  three  parts  A,  B 
and  C,  the  arrangement  and 
working  of  which  is  readily 
understood  from  the  illustra- 
tion. The  gas  enters  the  inflow 
tube  a',  and  passes  through  the 
opening  &,  into  the  interior  of  B. 
On  account  of  the  stopper  C  the 
gas  cannot  leave  except  by  pass- 
ing through  the  opening  c  into 
the  narrowed  portion  of  B.  It 
leaves  this  delivery  tube  at  the 
lower  end  (/)  and  through  the 
opening  d.  The  latter  supplies 
the  minimum  amount  of  gas 
necessary  to  keep  the  lamp 
burning.  This  supply  can  now 
be  regulated  with  absolute  pre- 
cision. The  opening  d  is  made 
through  the  parts  B  and  C.  By 
turning  C,  the  opening  through 
this  part  becomes  excentric  to 
that  of  B,  and  hence,  the  minimum  supply  of  gas  is  thus 
diminished. 

The  delivery  tube  /  should  not  be  too  narrow  and 
should  almost  touch  the  bottom  of  A.  The  first  droplet  of 

1  Centralblatt  fiir  Bakteriologle,  23,  p.  1054,  1898.  This  instru- 
ment can  be  obtained  of  Greiner  and  Friedrichs,  Stiitzerbach  i. 
Thuring-en. 


FIG.  37.    The  author's  thermoregulator. 


THE  INCUBATOR  AND  ACCESSORIES.  247 

mercury  as  it  issues  from  the  capillary  will  therefore  cut 
down  the  supply  of  gas.  The  instrument  is  thus  rendered 
more  sensitive. 

By  turning  part  B  the  inflow  of  gas  can  be  regulated. 
The  instrument,  therefore,  permits  regulation  of  the  max- 
imum inflow  and  minimum  outflow  of  gas.  Owing  to  the 
large  connecting  tubes  (a!  and  a")  it  is  possible  to  have  a 
large  supply  of  gas,  and  hence,  the  apparatus  can  be  used 
for  regulating  high,  as  well  as  relatively  low  tempera- 
tures. 

The  ordinary  regulator,  provided  with  a  narrow  cylin- 
der or  bulb  filled  with  mercury,  is  not  as  delicate  and  as 
responsive  as  might  be  desired.  This  is  due  to  the  fact 
that  the  volume  of  mercury  in  the  bulb  is  relatively  small 
and  hence  the  rise  and  fall  of  the  liquid  in  the  capillary, 
or  upper  tube  is  not  marked,  when  only  a  slight  variation  in 
the  temperature  occurs.  Consequently,  it  is  not  possible  to 
maintain  a  very  constant  temperature  with  a  plain  mercury 
regulator.  A  variation  of  one  or  two  degrees,  or  even 
more,  must  be  expected.  Moreover,  the  mercury  in  the 
side-arm,  since  it  is  exposed  to  the  air,  will  tend  to  counter- 
act the  action  of  the  remaining  mercury.  This  will  especially 
be  the  case,  when  there  is  a  marked  fluctuation  in  the  tem- 
perature of  the  room. 

The  most  delicate  regulator  is  that  which  contains  a 
large  volume  of  alcohol.  An  increase  of  a  fraction  of  a 
degree  in  the  temperature  will,  in  that  case,  cause  a  linear 
expansion  of  the  mercury  in  the  capillary  tube  which  will 
be  sufficient  to  shut  off  the  gas  supply.  This  fact  is  utilized 
in  the  construction  of  the  lower,  or  bulb  portion  (E)  of  the 
author's  regulator.  As  shown  in  Fig.  37,  the  capillary  tube 
is  prolonged  so  as  to  almost  touch  the  bottom  of  the  glass 
cylinder.  The  lower  portion  of  the  tube  is  provided  with 
a  small  bulb  which  serves  to  prevent  air  from  entering  the 
cylinder  when  at  any  time  the  regulator  is  disconnected. 


248  BACTERIOLOGY. 

Mercury  fills  the  capillary  and  covers  the  bottom  of   the 
cylinder  to  a  depth  of  about  1.5  cm. 

The  filling1  of  the  regulator  is  not  difficult.  For  this  purpose 
parts  B  and  C  are  removed,  and  the  end  of  A,  and  one  of  the  side 
arms  (a")  is  stoppered  air- tight.  The  other  arm,  or  gas  inflow  tube 
(a7),  is  connected  with  a  Chapman  aspirator.  The  rubber  tubing, 
leading  to  the  aspirator,  should  be  provided  with  a  clamp  or  stop- 
cock. The  adjusting  screw  D,  is  then  removed,  and  replaced  by  a 
short  rubber  tube  provided  with  a  clamp.  The  rubber  tube  connects 
with  a  short  narrow  glass  tube,  which  dips  into  a  beaker  of  absolute 
alcohol. 

The  pump  is  now  set  into  action.  The  clamp  on  the  regulating 
arm  D  is  slightly  opened,  till  the  alcohol  begins  to  pass  into  the 
tube,  after  which  it  is  closed.  When  the  air  has  been  evacuated,  as 
much  as  possible,  the  clamp  on  the  tubing  that  leads  to  the  pump  is 
closed,  while  that  on  regulating  arm  is  slowly  opened.  The  alcohol 
rapidly  fills  the  cylinder.  If  the  evacuation  is  incomplete,  the  regu- 
lator should  be  inverted,  and  the  air  again  exhausted,  after  which 
alcohol  can  be  admitted  as  before.  A  few  bubbles  of  air  that  may 
persist  can  be  disregarded,  inasmuch  as  they  will  be  absorbed  by  the 
alcohol. 

The  next  step  is  to  introduce  the  necessary  amount  of  mercury. 
For  this  purpose,  the  alcohol  bulb  of  the  regulator  is  immersed  in  a 
water-bath  which  has  been  heated  to  about  70°.  The  alcohol  expands 
and  fills  the  lower  portion  of  A.  When  A  has  been  nearly  half  filled 
with  the  expanded  alcohol,  mercury  is  then  added,  and  the  apparatus 
is  allowed  to  cool.  Additional  mercury  is  added,  if  need  be,  to  pre- 
vent the  entrance  of  air  into  the  cylinder.  The  regulating  arm  is 
likewise  filled  -with  mercury,  and  the  screw  inserted  about  one- 
half. 

At  ordinary  room  temperature  the  mercury  in  the  capillary 
tube  should  not  be  higher  than  the  regulating  arm.  The  ground  sur- 
faces of  the  regulator  should  be  lubricated  very  slightly  with  vaselin, 
or  better  with  a  mixture  of  bees-wax  and  olive  oil  (1  :  4).  The  inflow 
tube  a'  is  then  connected  with  a  gas-pressure  regulator,  or  with  the 
gas-supply  direct.  The  out-flow  tube  a"  is  connected  with  a  micro- 
burner.  A  suitable  cork,  or  rubber  stopper,  is  slipped  over  the  tube 
of  the  regulator,  which  is  then  placed  in  the  thermostat,  so  that  the 
bulb  is  immersed  in  the  water.  The  adjusting  screw  D  is  turned  out- 
ward, if  necessary,  so  as  to  allow  the  gas  to  escape  from  /.  The  gas 
is  turned  on  and  lighted.  As  the  temperature  rises  the  alcohol  ex- 


THE  INCUBATOR  AND  ACCESSORIES.  249 

pands  and  forces  up  the  mercury,  which  tends  to  close  up  /.  The 
adjusting-  screw  should  be  again  turned  outward,  if  need  be,  to  pre- 
vent this  from  happening-.  When  the  temperature  in  the  incubator 
has  reached  the  desired  point,  for  example,  37°,  the  adjusting-  screw 
should  be  turned  in  till  the  mercury  just  closes  the  opening-/,  of  the 
delivery  tube.  The  minimum  opening-  now  supplies  the  gas  to  the 
burner.  This  minimum  supply  should  now  be  reduced,  as  much  as 
possible,  by  turning-  C.  without  causing-  the  flame  to  shoot  down  inside 
of  the  burner.  As  soon  as  the  temperature  drops,  the  mercury  con- 
tracts, and  thus  allows  g-as  to  escape  from/.  In  a  few  minutes  the 
increase  of  heat  causes  the  mercury  to  again  shut  off  the  main  supply 
of  gas. 

Inasmuch  as  alcohol  boils  at  78°,  the  alcohol  regulator  should 
not  be  used  to  maintain  a  temperature  above  70°.  For  temperatures 
above  this  point  the  bulb  should  be  filled  entirely  with  mercury,  or 
the  alcohol  may  be  replaced  by  air  or  by  anilin,  which  boils  at  185°. 
The  ordinary  adjusting  screw  cannot  be  used  when  a  high  tempera- 
ture is  to  be  maintained.  For  such  purposes  the  regulator  should  be 
provided  with  a  glass  stop-cock,  which  connects  with  a  small  cup 
filled  with  mercury  (Fig.  37  D').  When  the  gas-supply  is  turned  off, 
as  in  the  case  of  a  water-bath  or  oven,  this  stop-cock  should  be 
opened. 

The  adjustment  of  the  ordinary  mercury  regulator  is  similar 
to  that  just  mentioned.  Frequently,  the  thread  of  mercury  is  broken 
and  air  is  thus  introduced  into  the  capillary  tube.  This  can  be  re- 
moved by  inserting  a  thin  platinum  wire,  or  a  drawn  out  capillary 
glass-tube,  and  gently  turning  it  about.  Care  should  always  be  taken 
to  remove  the  air  that  may  be  present  in  the  arm  D. 

Obviously,  a  continual  fluctuation  in  the  temperature 
of  the  incubator  will  exist.  A  good  alcohol  regulator, 
however,  should  not  allow  a  variation  of  more  than  one  or 
two-tenths  of  a  degree.  The  greater  the  volume  of  water 
in  an  incubator  the  more  constant  will  be  the  temperature. 
The  latter,  in  that  case,  will  be  likewise  more  independent 
of  any  variation  in  the  pressure  of  the  gas.  Frequently, 
the  pressure  of  the  gas  is  so  variable  that  it  is  not  possible 
to  maintain  a  perfectly  constant  temperature  in  an  incubator, 
even  when  this  is  provided  with  the  best  form  of  regulator. 
This  difficulty  can  be  obviated  by  employing  a  gas-pressure 
regulator. 


250 


BACTERIOLOGY. 


Gas-pressure  regulator. — The  apparatus  shown  in  Fig".  38 
was  designed  by  Murrill  '  and  is  used  in  this  laboratory.  In 
cheapness,  simplicity  and  effectiveness  it  excels  any  of  the 
ordinary  gas-pressure  regulators. 

As  indicated,  the  gas  enters  through  a  valve  which  is 
connected  with  a  gas  cylinder  or  float.  The  gas  passes 
into  the  interior  and  raises  this  cylinder. 
The  greater  the  pressure  of  gas  the 
higher  will  the  cylinder  rise.  As  it  does 
so,  it  turns  off  the  valve,  more  or  less 
completely.  The  cylinder  then  falls,  and 
hence  gas  is  readmitted.  The  pressure  at 
which  the  gas  is  delivered  can  be  regu- 
lated at  will  by  placing  suitable  weights 
on  the  cylinder.  In  this  laboratory,  the 
float  is  weighted  till  the  gas  is  delivered 
at  a  pressure  of  40  mm.,  as  observed  in  a 


FIG.  38.    Murrill's  gas-pressure  regulator. 


U  tube  containing  water.  The  cylinder  may  be  floated  in 
water,  but,  inasmuch  as  this  is  liable  to  evaporate,  it  is 
better  to  use  liquid  paraffin.  The  apparatus  is  provided 
with  two  outflow  tubes,  which  are  to  be  connected  with 
thermo-regulators.  It  is  of  convenient  size,  being  only  6 


1  Journ.  of   Applied  Microscopy  1,  p.  92,  1898. 
Bakteriologie  23,  p.  1056,  1898. 


Centralblatt  fur 


THE  INCUBATOR  AND  ACCESSORIES. 


251 


inches  in  diameter.     By  means  of  the  gas-pressure  and  tem- 
perature regulators,  which  have  been  described,  it  is  pos- 
sible to  maintain  a  constant  warmth  even  if  the  incubator- 
is  placed  in  a  room  where  the  ambient  temperature  is  liable 
to  considerable  variation. 


Micro-burners. — The  outflow  tube  of  the  thermo- 
regulator  should  be  connected  with  a  suitable  burner  placed 
underneath  the  incubator.  An  ordinary  Bunsen  burner  may 
be  used  in  some  instances,  but,  as  a  rule,  it  gives  off  too- 
much  heat,  and,  moreover,  the  flame 
is  liable  to  "shoot  back"  when  the 
supply  of  gas  is  low.  A  very  good 
micro-burner  can  be  obtained  by  un- 
screwing the  tube  of  the  Bunsen 
burner.  The  gas  now  burns  with  a 
luminous  flame,  but  this  does  not  in- 
terfere with  its  usefulness.  A  cyl- 
inder of  copper  wire-gauze  may  be 
placed  around  the  flame  to  prevent 
currents  of  air  from  extinguishing  it. 

The  small  narrow  Bunsen  burner, 
so-called  microburner,  can  be  used  to 

. ,  . ,  -, .  FIG.  39.   Koch's  safety  burner. 

better  advantage  than  the  ordinary  a— Metal  spiral,  one  on  each  side 

of  flame;    b — Arm  moved  by  the 

form.         Small     let,     luminOUS     micro-    spirals;    £- Weighted  lever  arm 

of  stop-cock,  supported  by  b. 

burners  can  also  be  used,  but  they 

possess  no   advantage   over  that  obtained  in  the  manner 

described  above. 

The  lamp  usually  employed  under  an  incubator  is 
known  as  the  safety  burner.  This  is  intended  to  shut  off  the 
flow  of  gas,  if  for  any  reason  the  light  should  go  out.  The 
gas  is  thus  prevented  from  escaping  into  the  room,  and 
danger  of  subsequent  explosion  is  thus  avoided.  Several 
forms  of  the  safety  burner  have  been  devised,  but  the  one 
which  is  used  perhaps  the  most  often  is  that  of  Koch. 
Obviously,  the  safest  procedure  is  to  place  the  incubator,  or 


252  BACTERIOLOGY 

other  apparatus  which  is  heated  day  and  night,  within  a 
good  hood. 

The  Koch  safety  burner  is  shown  in  Fig.  39.  It  is  a 
small  Bunsen  burner  provided  with  two  steel  springs. 
These  are  heated  by  the  flame  and  expand.  The  motion, 
thus  induced,  is  communicated  to  a  small  arm  which  can  be 
set,  so  that,  when  the  burner  is  lighted,  it  just  supports  the 
weighted  lever.  The  latter  is  connected  with  a  valve. 
When  the  light  goes  out  the  springs  cool  and  draw  the 
small  arm  from  under  the  weighted  lever.  This  then  falls 
and  shuts  off  the  flow  of  gas.  The  tube  of  the  Koch 
burner  is  not  infrequently  wider  than  it  should  be,  because 
the  minimum  supply  of  gas  from  the  thermo-regulator  is  too 
small  to  keep  the  light  burning  above  the  burner.  In  that 
case,  the  flame  drops  down  into  the  tube.  This  defect  can 
be  easily  remedied  by  inserting  a  short  piece  ( ^  in. )  of  brass 
tubing  just  inside  of  the  end  of  the  burner.  The  flame  is 
thus  made  narrower  and  is,  therefore,  less  liable  to  drop 
down. 

Thermometer. — Any  kind  of  a  glass  thermometer  can  be 
used  in  connection  with  a  thermostat,  but  it  is  preferable 
to  have  one  which  would  indicate  fractions  of  a  degree. 
The  thermometer  usually  employed  is  graduated  from 
0  to  60°  and  reads  tenths  of  a  degree.  A  Kappeler  maximum 
and  minimum  thermometer  is,  at  times,  very  useful  to  ascer- 
tain the  limits  of  variation  in  the  temperature. 


CHAPTER  X. 


RELATION    OF   BACTERIA  TO   DISEASE.     METHODS  OF    INFEC- 
TION AND  EXAMINATION. 


By  the  application  of  the  gelatin  plate  method,  or  its 
several  modifications,  it  is  possible  to  readily  separate  a 
given  organism  from  the  other  forms  which  may  be  present, 
and  to  obtain  thus  a  pure  culture.  The  isolated  colony  as 
it  develops  on  a  plate,  furnishes  the  first  pure  cultivation, 
since  it  is  derived  from  a  single  micro-organism.  Trans- 
plantations made  from  a  colony,  if  made  with  proper  pre- 
cautions, in  turn  yield  pure  cultures,  or  growths  contain- 
ing but  a  single  species.  Tube  cultures  can  thus  be  made 
in  gelatin,  bouillon,  agar,  blood-serum,  potato,  etc.,  and 
where  it  is  desired,  as  in  the  study  of  the  chemical  products 
of  bacteria,  flask  cultures  can  be  made. 

Relation  of  Bacteria  to  Disease. 

It  is  evident  that  in  order  to  demonstrate  that  a  given 
bacterium  is  the  cause  of  a  certain  fermentation,  or  of  the 
production  of  a  pigment,  or  of  phosphorescence,  etc.,  it  is 
necessary  that  it  should,  firsl,  be  isolated  and  obtained  in 
a  pure  culture.  And  that,  second,  a  pure  culture  of  the 
organism  grown  under  the  same  or  under  similar  condi- 
tions, should  give  rise  to  the  original  phenomenon — the 
production  of  the  same  fermentation,  pigment,  phosphor- 
escence, etc.  Having  thus  demonstrated  that  a  given  or- 
ganism is  the  cause  of  a  certain  change,  it  does  not  follow 
that  this  organism  has  the  exclusive  power  to  do  this. 


254  BACTERIOLOGY. 

Thus,  in  alcoholic  fermentation,  the  yeast  plant  is  com- 
monly said  to  be  the  cause,  but,  as  pointed  out  on  p.  96,  a 
large  number  of  different  species  of  yeasts  are  known 
which  have  this  power.  Moreover,  many  bacteria  and 
moulds  possess  similar  properties.  Again,  a  considerable 
number  of  bacteria  have  been  shown  to  be  capable  of  in- 
ducing acetic,  lactic,  butyric  acid  fermentations,  the  am- 
moniacal  and  hydrogen  sulphide  fermentations  of  urine,  the 
phosphorescence  of  sea-water,  etc. 

The  most  that  can  be  said  of  a  given  organism  which 
Induces  a  certain  change,  therefore,  is  that  it  is  the  cause 
in  that  particular  instance.  The  possibility  of  other  or- 
ganisms giving  rise  to  the  same  change,  or  effect,  or  to  the 
same  chemical  products  must  be  conceded,  and  the  demon- 
stration of  the  relation  of  an  organism  to  such  a  change 
rests  with  the  proof  that  it  is  a  cause. 

Just  as  there  are  organisms  which  induce  changes  in 
•dead  animal  or  vegetable  matter,  there  are  others  which 
are  capable  of  inducing  similar  changes  in  living  animals 
-and  plants,  thus  living  at  the  expense,  and,  frequently,  to 
the  detriment  of  the  host.  The  infectious  diseases  in  man, 
animals  and  plants,  possess  as  an  essential  characteristic 
the  property  of  transmissibility.  They  are  the  result:  first, 
of  infection  that  is  the  entrance  of  a  specific  micro-organ- 
ism; and  second,  of  intoxication  due  to  the  poisonous  pro- 
ducts elaborated  by  that  micro-organism. 

The  poisonous  chemical  compounds,  which  are  thus 
made,  may  produce  the  symptoms  and  the  changes  observed 
in  the  infectious  disease.  They  are  the  cause  of  those  symp- 
toms and  changes,  in  other  words,  of  the  resulting  intoxica- 
tion. But,  they  are  not  the  cause  of  the  disease  itself,  since 
the  symptoms  and  changes  thus  obtained,  are  not  transmissi- 
ble from  one  individual  to  another.  Chemical  substances 
have  no  power  of  multiplication  and  the  effect  observed  is, 
therefore,  directly  proportional  to  the  amount  of  the  chem- 
ical compound  introduced.  Micro-organisms,  however, 


RELATION  OF  BACTERIA  TO  DISEASE.  255 

have  the  power  of  multiplication,  and  the  introduction  of 
.a  minute  amount,  even  a  single  cell,  may  bring  about  en- 
tirely disproportionate  results.  The  invading-  organism  is, 
therefore,  the  cause  of  the  disease,  since  it  imparts  the 
characteristic  property  of  transmissibility ',  and,  through  the 
.action  of  its  chemical  products,  produces  the  symptoms 
and  effects  of  that  disease. 

In  order  to  positively  demonstrate  the  causal  relation 
of  a  micro-organism  to  a  given  disease,  it  is  necessary  to 
meet  the  following  requirements,  commonly  known  as  the 
four  rules  of  Koch: 

1. — The  organism  must  be  present  in  all  cases  of  that 
disease. 

2. — The  organism  must  be  isolated  and  obtained  as  an 
absolutely  pure  culture. 

3. — The  pure  culture  of  the  organism  when  introduced 
into  susceptible  animals  must  produce  the  disease. 

4. — In  the  disease  thus  produced,  the  organism  must 
be  found  distributed  the  same  as  in  the  natural  disease. 

To  these  four  requirements,  a  fifth  may  be  added, 
namely:  That  the  chemical  products  of  the  organism  must 
produce  the  characteristic  symptoms  and  effects  of  that 
disease. 

The  demonstration  of  the  constant  presence  of  an  or- 
ganism in  a  disease  is  accomplished  by  making  hanging- 
drop  examinations  of  the  fluids  or  exudates  of  the  body; 
by  making  stained  cover-glass  preparations,  or  by  stain- 
ing sections  of  the  tissues  and  organs.  Frequently,  the 
direct  detection  of  the  organism  is  difficult,  owing  either 
to  its  scarcity,  or  to  the  absence  of  definite  character- 
istics. In  such  cases  artificial  culture,  or  the  animal  ex- 
periment, will  usually  prove  the  presence  of  the  organ- 
ism. In  the  advanced  stages  of  some  diseases  the  germs 


256  BACTERIOLOGY. 

may  be  supplanted  or  outgrown  by  other  organisms — second- 
ary infection. 

The  value  of  artificial  culture,  in  suspected  cases  of 
disease,  is  seen  in  diphtheria.  On  the  other  hand,  the  pus, 
sputum,  or  pleuritic  fluid  from  a  diseased  person  may  not 
show,  on  direct  microscopic  examination,  any  sign  of  the 
tubercle  bacillus.  Yet,  if  such  material  is  injected  into  a 
guinea-pig,  it  may  produce  tuberculosis  and  death.  The 
microscope  failed  to  detect  the  few  bacilli  present  but  the 
living  body  afforded  these  same  cells  an  excellent  medium 
for  growth,  and,  as  a  result,  they  rapidly  multiplied  and 
eventually  brought  about  the  death  of  that  animal.  An 
examination  of  the  altered  tissue,  in  such  an  animal,  will 
reveal  the  presence  of  myriads  of  tubercle  bacilli.  Since 
living  cells  are  derived  only  from  cells  of  their  own  kind, 
it  follows  that  these  organisms  were  present  in  the  original 
diseased  condition. 

The  mere  fact  that  an  organism  is  constantly  present 
in  a  given  disease,  does  not  prove  that  it  is  the  cause. 
It  certainly  is  strong  presumptive  evidence  that  the 
organism  does  bear  a  causal  relation,  but  at  the  same 
time  the  possibility  must  be  admitted  that  it  may  be  an 
accompaniment,  or  even  a  consequent  of  that  disease. 
The  latter  view,  it  is  scarcely  necessary  to  note,  would  im- 
ply the  existence  of  spontaneous  generation.  To  complete 
the  chain  of  evidence,  it  is  necessary,  therefore,  to  obtain 
the  organism  in  an  absolutely  pure  culture,  free  from  any 
other  organism  or  foreign  chemical  poison.  Subsequent 
inoculation  of  susceptible  animals  with  such  cultures  must 
reproduce  the  disease.  Whenever  possible,  an  animal 
should  be  selected  which  is  naturally  susceptible  to  the 
disease.  An  animal  that  is  not  subject  to  the  disease  can 
hardly  be  expected  to  reproduce  the  typical  affection  on  in- 
oculation with  the  organism. 

The  isolation  of  the  organism  and  the  preparation  of 
pure  cultures,  is  accomplished  by  means  of  the  gelatin 


RELATION  OF   BACTERIA  TO  DISEASE.  257 

plate  method,  or  by  its  modifications.  The  isolated  colony 
which  develops  on  a  plate,  is  derived  from  a  single  cell 
and  is,  therefore,  a  pure  culture.  Transplantations  frorr^ 
such  a  colony,  when  properly  made  into  tubes  of  gelatin, 
agar,  or  bouillon,  in  turn  give  rise  to  pure  cultures.  Sub- 
sequent transplantations,  from  tube  to  tube,  can  be  made  as 
often  as  may  be  desired,  or  as  may  be  necessary.  Each 
growth  thus  obtained  is  called  a  generation. 

In  many  cases,  as  in  tuberculosis,  anthrax,  and  hog  chol- 
era, the  organisms  have  thus  been  carried  through  several 
hundred  consecutive  generations  without  impairment  of 
pathogenic  properties.  In  other  instances,  as  in  glanders, 
the  organism  does  not  find  in  our  artificial  media  the  condi- 
tions favorable  for  its  growth,  and,  as  a  result,  it  under- 
goes a  physiological  alteration  so  that  the  cultures  become 
less  and  less  active,  till  finally  they  cease  to  have  any  effect 
on  animals.  This  change  in  the  physiological  properties  of 
an  organism — known  as  attenuation — is  frequently  accom- 
panied by  a  corresponding  decrease  in  the  vitality  of  the 
growth,  so  that,  when  the  virulence  is  wholly  lost,  the  cul- 
ture soon  dies  out.  Sometimes,  however,  the  organism 
adapts  itself  to  the  artificial  media,  and  continues  to  grow, 
although  with  diminished  pathogenic  properties.  The 
phenomenon  of  attenuation,  it  should  be  understood,  is 
not  confined  to  the  pathogenic  bacteria.  Corresponding 
changes  in  the  activities  of  many  non-pathogenic  bacteria 
are  constantly  being  met  with.  Thus,  a  phosphorescing 
bacillus  may  emit  a  strong  light  when  first  obtained,  but  in 
a  short  time  it  may  lose  this  power.  The  B.  ruber 
of  Kiel  usually  give  an  intense  red  growth  on  potato,  but 
it  is  not  uncommon  to  meet  with  it  as  a  colorless  growth. 
Again,  a  given  -organism  may  rapidly  coagulate  milk,  but 
after  it  has  been  cultivated  on  artificial  media  it  may  lose, 
more  or  less  completely,  the  power  of  altering  the  compo- 
sition of  milk.  These  illustrations  teach,  that  when  bac- 

17 


258  BACTERIOLOGY. 

teria  are  under  the  very  best  conditions  of  environment 
they  possess  a  maximum  physiological  activity.  They 
may  give  rise  to  intense  poisons,  pronounced  pigments,  or 
other  characteristic  chemical  products.  When,  however, 
these  conditions  are  altered  and  the  environment  becomes 
unfavorable,  the  bacteria  are  likely  to  respond  at  once  by 
a  corresponding1  lessening1  of  vitality.  The  chemical  pro- 
ducts which  were  formed,  when  the  organism  was  in  a 
vigorous,  healthy  condition,  are  now  diminished  in  amount 
and  may  even  be  replaced  by  others.  Attenuation,  there- 
fore, is  a  phenomenon  which  is  common  to  all  bacteria, 
although  the  term  was  first  employed  in  connection  with 
the  loss  of  virulence  of  certain  pathogenic  organisms. 

The  above  four  rules  have  been  fully  complied  with  in 
a  large  number  of  infectious  diseases.  In  others  the  first 
two  rules  are  satisfied  but  the  third  is  not,  owing-  to  the 
difficulty  of  obtaining  a  susceptible  animal.  Again,  the 
first  rule  may  be  the  only  one  complied  with,  as  in  leprosy, 
where  the  isolation  of  the  organism  has  not,  thus  far, 
been  unquestionably  successful.  And  again,  a  large  num- 
ber of  infectious  diseases  remain  (such  as  small-pox,  scar- 
let fever  and  measles)  in  which  even  the  presence  of  a 
specific  organism  has  not  been  definitely  shown. 

It  is  evident  that  while  a  great  deal  of  information  has 
been  gained  during  the  past  15  years  in  regard  to  the  caus- 
ation of  disease,  there  still  remains  a  great  deal  of  work 
to  be  done.  Enough  facts  have  been  gathered  to  clearly 
demonstrate  the  importance  of  the  new  science  of  bacteri- 
ology to  medicine,  surgery  and  hygiene.  What  was  once 
called  the  "germ  theory  of  disease"  has  ceased  to  be  a 
theory  since  it  has  been  reduced  to  incontrovertible  facts. 
That  micro-organisms  are  the  causes  of  disease  has  been 
demonstrated. 

Although  many  of  the  infectious  diseases  have  been 
shown  to  be  due  to  bacteria,  it  must  not  be  forgotten  that 


RELATION   OF  BACTERIA  TO  DISEASE.  259 

other  low  forms  of  plant  and  animal  life  possess  similar 
properties. 

Certain  fungi  are  known  to  live  as  parasites   on   the_ 
higher  animals.     Thus,  some  skin  diseases  of  man  are  due 
to  this  class  of  organisms.     These  may  be  grouped  together 
under  the  head  of  fungous  diseases. 

A^ain,  certain  uni-cellular  animal  organisms,  belonging 
to  the  group  of  protozoa,  invade  the  body  and  give  rise  to 
disease.  Malaria  is  due  to  one  of  these  animal  parasites. 
The  diseases  due  to  these  organisms  can  be  designated  as 
protozoal. 

The  bacterial,  fungous  and  protozoal  affections  are 
included  in  the  large  group  of  infectious  or  microbic  diseases. 
It  has  been  stated  that  the  cause  of  certain  diseases,  like 
measles  and  scarlet  fever,  is  as  yet  wholly  unknown.  The 
cause,  when  discovered,  may  belong  to  one  of  these  three 
groups,  but  not  necessarily  so.  It  is  customary  to  consider 
the  bacterial  cell  as  the  smallest  living  mass  of  matter.  It 
is  difficult,  indeed,  to  conceive  of  the  existence  of  organized 
beings  smaller  than  the  " infinitely  small  bacteria."  And 
yet,  the  fact  that  all  known  methods  of  study  have  failed 
to  discover  the  cause  of  certain  diseases  would  indicate 
that  we  must  look  to  an  undiscovered  group  of  microbes 
which  must  perforce  be  smaller  than  any  known  bacteria. 

The  first  step  has  already  been  taken  in  the  direction 
of  opening  up  this  new  field  of  study.  By  means  of  the 
collodium  sac  method,  which  will  be  described  in  Chapter 
XIV. ,  it  has  been  shown  that  the  cause  of  pleuro-pneumonia 
in  cattle  is  an  organism  so  small,  that,  with  the  best  micro- 
scope and  a  magnification  of  2000  diameters,  it  is  still 
impossible  to  make  out  its  form.  Evidently,  this  is  the 
first  of  a  new  group  of  microbes. 

Pathogenic  micro-organisms  are  of  first  importance  in 
so  far  as  they  produce  disease  in  man  and  in  the  lower 
animals.  The  fact,  however,  should  not  be  lost  sight  of 
that  similar  organisms  produce  disease  in  plants.  A  large 


260  BACTERIOLOGY. 

number  of  bacterial  and  fungous  diseases  of  plants  are 
known  at  the  present  day.  The  various  species  of  the 
vegetable  kingdom,  just  as  man  and  the  lower  animals, 
are  subject  to  their  peculiar  infectious  diseases. 

Methods  of  Infection. 


As  indicated  in  the  preceding  part,  the  detection  of 
an  organism  in  every  case  of  a  given  disease  does  not,  in 
itself,  constitute  a  proof  that  it  is  the  cause  of  that  disease. 
It  is  necessary  to  carry  the  investigation  farther  in  order  to 
furnish  an  indubitable  proof.  The  second  rule  of  Koch 
requires  that  the  suspected  organism  shall  be  isolated,  in 
other  words,  obtained  in  a  condition  of  absolute  purity.  If 
the  pure  culture  of  the  suspected  organism,  isolated  accord- 
ing to  the  methods  heretofore  described,  on  inoculation  into 
susceptible  animals  reproduces  the  characteristic  symptoms 
and  pathological  lesions  of  that  disease  there  can  be  no 
escape  from  the  conclusion,  that  the  organism  tested  is  the 
causal  factor  of  the  disease  in  question. 

Extreme  care  must  be  taken  in  order  to  obtain  an  abso- 
lutely pure  culture.  Likewise,  the  utmost  care  must  be 
employed,  when  making  the  inoculations  of  animals,  to 
prevent  the  accidental  introduction  of  foreign  organisms. 
•Careless  inoculation  may  give  wholly  misleading  results. 
All  operations  on  animals,  even  the  most  trivial,  should  be 
carried  out  under  as  nearly  sterile  conditions  as  possible. 
Every  instrument  employed  should  be  sterile,  and,  while  it 
is  not  possible  to  render  the  hands  of  the  operator  and  the 
skin  of  the  animal  as  free  from  organisms  as  in  the  case  of 
an  instrument,  yet  nothing  should  be  left  undone  to  decrease 
the  number  of  bacteria  present  and  to  lessen  thus  the  danger 
•of  mixed  infection. 

The  experimental  methods  of  infection  are .  numerous. 
;Some  are  exceedingly  simple,  whereas  others  entail  a  more 


METHODS    OF    INFECTION.  261 

or  less  difficult  surgical  operation.  They  include,  moreover, 
all  the  known  avenues  of  infection  whereby  man  and 
animals  contract  disease.  The  several  methods  employed 
will  now  be  described  as  briefly  as  possible.  The  guinea- 
pig,  rabbit  and  white  mouse  are  the  animals  employed  most 
often.  The  white  rat,  house  mouse,  pigeon,  chicken,  dog 
and  others  are  used  less  frequently. 

1. — Cutaneous  application. — Bacteria  cannot  penetrate 
into  the  interior  of  the  body  through  an  unbroken  skin  or 
mucous  membrane.  Nevertheless,  a  person  may  become 
infected,  without  any  visible  injury,  as  sometimes  occurs 
when  making  operations  on  the  living,  or  when  making 
post-mortem  examinations  of  the  dead.  In  such  cases,  the 
existence  of  microscopic  abrasions  or  fissures,  or  an  uncon- 
scious puncture  is  looked  upon  as  an  explanation  of  the 
infection.  The  organism  to  be  tested  in  this  manner  is 
rubbed  into  the  skin  as  much  as  possible  with  the  aid  of 
some  fat  or  vaselin.  In  the  study  of  pus-producing  organ- 
isms results  have  been  obtained  by  this  method  which  are 
strictly  analogous  to  conditions  observed  in  man.  The 
production  of  boils  or  felons  has  thus  been  brought  about 
experimentally. 

2. — Subcutaneous  application  or  injection. — By  application 
is  meant,  in  this  case,  the  introduction  of  the  organism 
through  an  opening  in  the  skin:  This  is  made  by  means  of 
a  lance,  or  pair  of  scissors.  Through  the  small  nick,  thus 
made,  the  platinum  wire  or  other  instrument,  laden  with 
the  organism,  is  inserted  into  the  subcutis  and  the  material 
is  spread  about  as  much  as  possible.  This  method  may  be 
considered  as  analogous  to  the  ordinary  wound  infection  in 
man  and  animals.  A  puncture  with  a  sharp  piece  of  wood, 
glass  or  metal  enables  the  introduction  of  the  organisms 
that  may  be  present  on  such  a  surface.  Tetanus,  or  lock- 
jaw, is  a  striking  illustration  of  this  method  of  infection. 


BACTERIOLOGY. 


In  the  above  procedure,  the  hair  is  removed  from  over  the  desired 
area  by  means  of  scissors.  The  spot  is  then  rubbed  thoroughly,  with 
a  cloth 'or  sponge  soaked  in  mercuric  chloride  (1-1000)  or  lysol  (2  per 
cent.).  This  skin  is  then  raised  by  the  fingers,  or  with  forceps,  and 
the  small  nick  or  incision  is  made.  This  should  extend  through  the 
skin.  It  is  advisable  to  insert  the  lance  or  a  blade  of  the  scissors 
under  the  skin,  thus  making  a  pouch.  Guinea-pigs  and  rabbits  are 
inoculated  over  the  side.  The  animal  is  held  down  on  the  table 
by  an  assistant.  Mice  and  rats  are  inoculated  over  the  root  of  the 
tail.  The  tail  of  the  animal  is  seized  with  a  pair  of  rat  or  crucible 
forceps  (Fig.  46  b,  p.  273)  and  drawn  over  the  edge  of  the  jar.  The 
weighted  wire-gauze  cover,  of  the  battery  jar  in  which  it  is  confined, 
is  moved  slightly  to  one  side  so  as  to  allow  the  tail  and  lower  portion 
of  the  body  to  be  drawn  out.  The  tail  is  held  by  the  left  hand  while 
the  opening  and  subsequent  inoculation  are  made  with  the  right. 

Subcutaneous  injection  is  practised  by  means  of  a 
sterile  syringe.  For  this  purpose  the  Koch  syringe  has 
been  used  extensively,  but  it  cannot  be 
said  to  possess  any  marked  advantage 
over  a  good  hypodermic  syringe,  and, 
it  certainly  is  less  convenient.  It  is  ad- 
visable to  employ  a  syringe  with  an 
adjustable  rubber  piston.  The  stem  of 
the  piston  should  be  graduated  to  in- 
dicate c.c.,  and  should  have  a  set-screw, 
thus  enabling  the  operator  to  inject 
the  amount  desired  with  the  least 
trouble  or  uncertainty.  The  Roux- 
Collin  syringe,  or  one  such  as  is  shown 
in  Fig.  40,  is  the  most  durable  and 
useful  of  its  kind. 

The  syringe  should  be  sterilized  in 
boiling  water  in  a  water-bath.  An 
enamelled  stewing-pan  heated  by  a 
Fletcher  radial  burner  (Fig.  41,  a,  &)  is  an  extremely  useful 
accessory  in  the  laboratory.  The  syringe  should  not  be 
placed  direct  in  the  boiling  water.  .  The  latter  should  be 
drawn  up  into  the  syringe  and  expelled  several  times  in 


FIG.  40. 
inge.    F— 


Adjustable  syr- 
Rubber  piston. 


METHODS   OF  INFECTION. 


263 


succession.  In  this  way,  the  syringe  is  heated  up  evenly. 
It  is  then  partially  filled  with  water,  placed  on  the  syringe 
holder  (Fig.  41  c),  and  immersed  in  the  boiling  water.  An 
exposure  for  three  to  five  minutes  under  these  conditions 
will  be  sufficient.  The  holder  with  the  syringe  is  then  re- 
moved and  the  latter  is  allowed  to  cool,  after  which  the 
contained  water  is  expelled.  The  bacterial  fluid  is  now 
drawn  up  into  the  syringe  through  the  needle.  If  any  air 
is  present  this  should  be  expelled,  by  holding  the  syringe 
in  a  vertical  position  and  applying  gradual  pressure  to  the 
piston.  Before  doing  this,  it  is  advisable  to  pass  the 
needle  through  a  piece  of  sterile  filter  paper,  slipped  over 
the  end  of  a  wide  test-tube.  The  latter  receives  the  few 
drops  of  liquid  that  are  expelled  at  the  same  time  with  the 
air. 


FIG.  41.  a— Fletcher  radial  burner;  b— Enameled  stew-pan  or  water-bath;  c— Syr- 
inge holder;  ^—Syringe. 

After  using  the  syringe,  it  should  be  rinsed  with  boil- 
ing water  in  the  manner  mentioned,  and  then  immersed  in 
the  boiling  water  and  sterilized.  In  order  to  prevent  rust- 
ing of  the  needles,  these  should  be  kept  in  a  10  per  cent, 
solution  of  borax. 

When  it  is  desirable  to  inject  a  relatively  large  quan- 
tity of  liquid  into  an  animal,  or  to  inject  a  number  of  animals 
in  succession  with  the  same  liquid  (as  in  the  immunization 
of  horses),  then  an  arrangement  similar  to  that  shown  in 


264 


BACTERIOLOGY. 


Fig".  42  is  made  use  of.     This  apparatus  can  be  readily  im- 
provised out  of  an  ordinary  250  c.c.  measuring-  cylinder. 

Usually,  bouillon  cultures  are  employed  for  the  pur- 
pose of  injection.  Occasionally,  however,  the  bacterial 
growth  is  on  a  solid  medium,  like  ag-ar  or  potato.  In  that 
case,  bouillon  or  sterile  water  should  be  introduced  into  the 
tube  by  means  of  a  drawn  out  tube  pipette  (Fig-.  61).  The 

growth  is  then  thoroug-hly 
stirred  up  in  the  liquid  by 
means  of  the  end  of  the 
pipette.  The  suspension  is 
then  drawn  up  into  the  tube, 
and  transferred  to  a  sterile 
Esmarch  dish,  or  to  a  small 
sterile  conical  test-glass  (Fig-. 
43).  In  the  case  of  a  bouillon 
culture,  this  is  transferred 
directly  to  the  sterile  vessel. 
The  neck  of  the  tube,  after 
withdrawal  of  the  cotton 


FIG.  42.     Apparatus  for  injecting  large  quantities  of  liquid. 

plug-,  must,  of  course,  be  heated  in  the  flame  before  pouring- 
out  the  culture. 

When  injecting-  small  animals  care  must  be  taken  that 
the  needle  does  not  pass  throug-h  the  abdominal  wall  into 
the  peritoneal  cavity.  A  fold  of  the  skin  should  be  raised, 
and  the  needle  inserted  while  the  syring-e  is  held  in  a 
position  almost  parallel  to  that  of  the  body.  A  success- 
ful subcutaneous  injection  will  show  a  swelling-  over  the 
place  of  inoculation. 


The   injection  is  made  most  conveniently  over  the  abdominal 
wall,  inasmuch  as  the  skin  is  softer  there  than  elsewhere.    The  rabbit 


METHODS  OF   INFECTION.  265 

or  guinea-pig"  should  be  held  on  its  back  by  an  assistant.  The  head  and 
front  leg's  should  be  held  in  the  palm  of  the  left  hand,  while  the  hind 
leg's  are  held  down  firmly  with  the  right.  A  very  convenient  way  of 
holding-  a  rabbit  is  for  the  assistant  to  take  it  by  the  ears  and  hind 
legs  and  stretch  it  over  his  hip  or  knee. 

Guinea-pig's  may  be  inoculated  without  the  aid  of 
an  assistant  by  placing-  these  within  an  ordinary  large 
graduate  cylinder.  A  cylindrical  holder  of 
copper,  as  described  by  Voges,  is  preferable  to 
the  use  of  a  glass  one.  Fig.  44  illustrates  the 
manner  of  using  these  holders.  It  is  well  to 
have  2  or  3  of  these  cylinders  of  different  diam- 
eters, so  as  to  fit  guinea-pigs  of  various  size. 
The  length  should  be  about  18  to  20  cm.  The 
diameters  may  be  6  and  8  cm.,  respectively.  FIG. 43.  sterile 
The  longitudinal  slit  should  be  about  1  cm., 
wide  and  10  cm.  long.  Equally  useful  holders  can  be 
made  by  boring  holes,  of  the  size  given,  in  a  block  of 
wood. 

The  mouse  or  rat  is  usually  picked  up  by  the  tail  with  a  pair  of 
rat  forceps  (Fig.  46  6)  and  set  on  the  table.  A  folded  piece  of  cloth  is 
dropped  on  its  head,  which  is  then  seized  between  the  thumb  and  fin- 
ger. The  tail  and  hind  legs  are  held  by  the  other  hand.  When  hold- 
ing the  animal  thus,  the  pressure  should  be  applied  to  the  head  and 
not  to  the  neck  or  chest.  A  better  procedure  than  the  above,  which, 
moreover,  dispenses  with  the  need  of  an  assistant,  is  to  grasp  the  ani- 
mal by  the  nape  of  the  neck  in  a  Collin  pressure  forceps.  The  forceps 
are  now  suspended  from  a  hook  or  from  a  retort  stand,  and  the  tail 
and  hind  legs  grasped  in  the  fingers  of  the  left  hand.  The  animal  is 
thus  placed  on  a  slight  stretch  and  the  inoculation  is  made  with  the 
right  hand. 

3. — Intravenous  injection. — The  direct  introduction  into 
the  circulation  of  a  bacterial  suspension  or  toxin  is  fre- 
quently practised.  This  method  is  especially  valuable  in 
the  immunization  of  animals.  Inasmuch  as  the  liquid  is  in- 
troduced directly  into  the  blood  current,  and  is  thus  rapidly 
distributed  throughout  the  body,  it  follows  that  the  results 


266 


BACTERIOLOGY. 


obtained  by  this  procedure  are  more  rapid  and  are  far 
more  dangerous  than  a  subcutaneous  injection. 

The  injection  is  made  by  means  of  the  syringe  or  injec- 
tion apparatus,  described  above.  Special  care  must  be 

taken  to  see  that  all  air-bubbles 
are  expelled  from  the  syringe 
before  it  is  used.  Air  in  itself 
is  not  dangerous,  provided  it  is 
not  injected  rapidly  and  in  large 
bubbles.  It  is  well,  therefore, 
to  begin  the  injection  as  slowly 
as  possible. 

The  rabbit  is  the  animal 
employed  most  often  for  this 
method  of  injection.  This  is 
due  to  the  fact  that  the  mar- 
ginal branch  of  the  posterior 
auricular  vein  is  easily  entered 
with  the  needle.  With  a  little 
FIG.  44.  The  Voges  cylindrical  holder,  practice  the  operation  can  be 

for  guinea-pigs.  n  , 

done  almost  as  expeditiously  as 

an  ordinary  subcutaneous  injection.  It  is  well  to  select  an 
animal  with  white  ears,  and  in  which  the  marginal  vein  is 
fairly  large.  Occasionally  this  vein  is  very  narrow  and  it 
is  then  extremely  difficult  to  enter.  It  is  advisable,  more- 
over, to  use  a  sharp  new  needle. 

The  animal  is  placed  on  the  table  before  a  window.  The  assistant 
holds  the  animal  down  gently  but  firmly.  One  hand  rests  over  the  pel- 
vis, the  other  covers  the  head  and  holds  the  front  leg's.  There  is 
really  but  one  vein  in  the  rabbit's  ear  which  can  be  used  for  the  pur- 
pose of  injection.  The  large,  middle  veins  of  the  ear  are  imbedded  in 
loose  connective  tissue,  and  hence,  readily  roll  away  from  the  needle. 
The  posterior  marginal  vein  is  narrow,  but  it  is  imbedded  in  such  a 
manner  that  it  cannot  move  away.  Hence,  little  difficulty  is  experi- 
enced in  penetrating  this  vein  although  it  may  seem  to  be  narrower 
than  the  needle. 

By  means  of  a  pair  of  scissors,  preferably  bent,  the  hair  is  re- 
moved from  the  surface  over  this  marginal  vein.  A  piece  of  cotton, 


METHODS  OF  INFECTION.  267 

or  cloth,  dipped  in  warm  water,  is  then  rubbed  over  this  surface.  This 
usually  makes  the  vein  more  prominent.  A  small  clamp  can  be  ap- 
plied over  the  lower  part  of  the  vein  to  cause  it  to  distend,  but  this  is 
by  no  means  necessary.  The  ear  is  then  bent  over  the  index  fingerr 
thus  supporting-  that  portion  of  the  vein  which  it  is  desired  to  enter. 
The  syringe  is  held  almost  parallel  to  the  course  of  the  vein.  As  the 
needle  enters  the  tissue  one  meets  with  the  expected  resistance,  but 
the  moment  it  enters  the  vein,  this  resistance  disappears.  After  a 
very  few  trials,  the  student  is  able  to  tell  whether  or  not  the  needle 
has  entered  the  blood-vessel.  It  should  now  be  inserted  into  the  vein 
about  a  quarter  of  an  inch,  and  the  thumb  then  applied  over  the 
point  of  entrance.  The  needle  and  ear  are  thus  held  between  the 
thumb  and  forefinger.  Care  must  be  taken  to  hold  the  needle  par- 
allel to  the  course  of  the  vein.  The  liquid  can  now  be  gradually  in- 
jected, removing-  first  of  all  the  clamp  from  the  base  of  the  ear,  if 
one  has  been  used. 

As  the  liquid  flows  into  the  vein  it  drives  the  blood  before  it, 
and  hence,  only  a  colorless  stream  will  be  seen.  If  there  is  any  re- 
sistance to  the  pressure  on  the  piston,  and,  especially  if  a  swelling- 
forms  at  the  point  of  inoculation,  it  is  evident  that  the  vein  has  been 
missed.  The  needle  should  therefore  be  withdrawn,  and  the  attempt 
repeated  a  trifle  lower  down  the  vein.  It  is  well  always  to  make 
the  first  attempt  at  penetrating-  a  vein  as  high  up  as  possible. 
When  the  needle  is  withdrawn,  the  place  of  inoculation  should  be 
held  a  few  minutes  between  the  fingers,  or  a  clamp  should  be  applied 
to  prevent  hemorrhage. 

When  the  amount  of  the  liquid  to  be  injected  is  large, 
it  is  better  to  make  the  injection  in  that  case  into  the  jugu- 
lar vein.  This  method  requires  a  little  more  time,  and 
greater  precaution  than  the  preceding.  For  this  purpose 
the  animal  must  be  fastened  down  on  some  form  of  a  holder 
and  anesthetized. 

The  rabbit  holder  of  Czermak  is  frequently  employed 
for  fastening  down  rabbits  and  guinea-pigs.  The  Latapie 
holder  shown  in  Fig.  45,  is  admirably  adapted  for  laboratory 
purposes.  It  can  be  used  equally  well  for  rabbits,  guinea- 
pigs,  chicken  or  pigeons.  The  flexed  joint  is  placed  over 
the  cross-piece  of  the  clamp,  and  the  ring  is  then  bent  over 
till  it  is  held  by  a  spring  catch. 


268  BACTERIOLOGY. 

The  hair  is  removed  from  the  neck  by  means  of  a  pair  of  bent 
scissors.  The  part  should  then  be  shaven  clean  and  washed  with  an 
antiseptic.  A  median  incision  is  made  along-  the  neck  with  a  sterile 
scalpel,  and  the  skin  is  loosened  from  the  subcutis  by  using-  the  handle 
of  the  knife,  or  the  fing-ers.  The  external  jugular  vein  which  lies  on 
the  side,  covered  by  subcutaneous  tissue,  is  now  exposed  as  carefully 
as  possible.  The  size  of  the  vein  is  such  that  no  difficulty  will  be  ex- 
perienced in  finding-  or  entering-  it.  A  ligature  should  then  be  ap- 
plied to  the  vein  and  the  wound  in  the  skin,  sewed  up  as  neatly  as 
possible.  A  few  tufts  of  cotton  should  be  spread  over  the  incision, 
and  then  moistened  with  collodium.  When  this  has  dried  the  animal 
can  be  released. 


FIG,  45.    Latapie's  animal  holder. 

In  the  above  method  of  inoculation,  every  possible  pre- 
caution must  be  taken  to  prevent  accidental  infection. 
The  hands,  as  well  as  the  neck  of  the  animal,  must  be 
thoroughly  disinfected.  All  instruments,  ligatures,  etc., 
must  be  sterile.  The  instrument  sterilizing'  case,  shown  in 
Fig.  48,  p.  275,  is  used  for  this  purpose. 

Intravenous  injection  is  practised  frequently  during 
the  immunization  of  horses  against  diphtheria,  pest,  etc. 
The  operation  as  made  on  the  horse  is  relatively  as  easy  as 
the  injection  into  the  veins  of  the  ear  of  a  rabbit.  An  at- 
tendant should  compress  the  jugular  vein  with  the  thumb, 
while  the  operator  cuts  away  the  hair  over  the  vein.  This 
cut  area  is  then  thoroughly  rubbed  with  lysol,  or  some 
other  disinfectant.  The  skin  is  first  pierced  with  a  lance, 
and  then  the  trochar  is  inserted  as  nearly  parallel  to  the 
vein  as  possible.  After  withdrawal  of  the  trochar,  the 
opening  should  be  compressed  by  a  finger,  and  eventually 
it  may  be  coated  with  collodium. 


METHODS  OF  INFECTION.  269 

4. — Intra-peritoneal  injection.. — This  method  of  experi- 
mental infection  is  employed  as  frequently  as  the  subcu- 
taneous one  already  described.  When  organisms  are 
introduced  directly  into  the  peritoneal  cavity,  the  results 
of  infection  rapidly  follow.  The  animal  may  die  in  12 
hours  or  less.  Moreover,  it  should  be  noted  that  many 
bacteria,  which  will  not  kill  when  injected  subcutaneously, 
are  invariably  fatal  when  introduced  into  the  peritoneal 
cavity.  A  good  example  of  this  is  seen  in  the  colon 
bacillus. 

The  animal  to  be  injected  is  placed  on  its  back,  or,  in 
the  case  of  the  mouse  or  rat,  it  is  suspended  in  the  manner 
described  on  p.  265.  A  fold  of  the  skin  is  raised  and  the 
needle  introduced  at  an  angle  of  about  45  degrees.  The 
fact  that  the  needle  enters  the  peritoneal  cavity  is  recog- 
nized by  the  absence  of  resistance  to  the  needle  and  by  the 
absence,  at  the  point  of  inoculation,  of  a  local  swelling 
which  would  indicate  that  the  fluid  has  entered  the  subcu- 
taneous tissue.  As  a  rule,  there  is  very  little  or  no  danger 
of  injuring  the  intestines.  In  making  injections  it  is  advis- 
able to  cut  away  the  hair  over  the  point  of  inoculation, 
thus  marking  as  it  were,  the  place  of  operation.  .  It  is 
hardly  necessary,  as  a  rule,  to  attempt  a  thorough  disin- 
fection of  the  skin  previous  to  making  an  injection. 

Intra-peritoneal  injection  into  a  large  animal,  as  the 
horse,  is  made  by  means  of  a  trochar.  The  animal  is  held 
in  the  standing  position  and  the  trochar  introduced  through 
the  abdominal  wall,  after  previously  cutting  the  skin 
with  a  lance.  A  point  4  or  5  inches  in  advance  of  the 
anterior  iliac  spine  is  selected  as  the  site  of  inoculation. 

In  certain  cases  it  is  desirable  to  observe  the  disease 
process  in  the  peritoneal  cavity,  or  to  introduce  solid 
masses  into  this  cavity.  In  that  event  it  is  necessary  to 
make  a  laparotomy,  in  which  case  the  utmost  precaution 
to  prevent  accidental  infection  must  be  observed.  This 
operation  is  resorted  to  whenever  the  collodium  sac  method 


270  BACTERIOLOGY. 

is  employed.     The  necessary  directions  will  be.  found  under 
that  head  (Chapter  XIV). 

5. — Intra-pleural  injection. — This  method  is  sometimes 
employed,  though  rarely.  Great  care  must  be  taken  to 
prevent  injury  to  the  lung's  or  heart.  The  introduction  of  a 
large  quantity  of  liquid  is  in  itself  dangerous.  The  needle 
is  introduced  into  the  right  pleural  cavity  at  some  distance 
from  the  median  line.  The  results  of  infection  are  brought 
on  rapidly,  as  in  the  case  of  intraperitoneal  injection. 

6. — Infection  of  the  anterior  chamber  of  the  eye. — This 
method  has  been  followed  with  excellent  results  in  the 
study  of  tuberculosis.  'The  pathological  changes  induced 
can  be  observed  from  day  to  day.  The  operation  itself  is 
simple.  The  eye  is  first  anesthetized  with  cocain.  An 
incision  is  made,  by  means  of  a  very  narrow  scalpel,  at  the 
juncture  of  the  cornea  and  sclera.  Through  the  opening 
thus  made,  the  material  to  be  tested  is  inserted  into  the 
anterior  chamber.  Instead  of  making  an  incision  the  bac- 
terial fluid  can  be  injected  by  means  of  a  syringe. 

7. — Infection  of  the  lymphatics. — This  is  accomplished 
easily  by  injecting  directly  into  a  testicle.  The  infection 
spreads  rapidly  through  the  neighboring  lymphatics  and  as 
a  result  marked  alterations  result  in  a  short  time. 

8. — Intra-cranial  injection. — This  is  the  method  employed 
by  Pasteur  and  his  pupils  in  producing  rabies  in  rabbits 
and  other  animals.  The  procedure  will  be  described  in 
Chapter  XIV. 

9. — Infection  along  the  respiratory  tract.—  This  may  be 
brought  about  (1)  by  the  inhalation  of  the  finely  divided 
organisms;  (2)  by  injection  into  the  trachea.  The  former 
method  is  analogous  to  the  usual  mode  of  infection  in 


METHODS  OF  INFECTION.  271 

tuberculosis.  An  atomizer  is  employed  to  bring  the  organ- 
isms into  a  finely  divided  condition.  It  need  hardly  be 
stated  that  this  method  is  extremely  dangerous  to  the_ 
operator.  In  fact,  it  is  the  most  dangerous  procedure  in  the 
entire  bacteriological  technique,  and,  for  that  reason,  it  is 
but  rarely  resorted  to. 

The  intra-tracheal  injections  have  been  extremely  valu- 
able in  the  study  of  diphtheria.  The  method  is  simple,  and 
is  carried  out  in  much  the  same  way  as  when  making  injec- 
tions into  the  jugular  vein.  The  animal  is  fixed  on  its  back 
and  anesthetized.  After  removal  of  the  hair,  and  after 
local  disinfection,  a  median  incision  is  made.  The  trachea 
is  then  gradually  exposed,  making  use  of  a  grooved  sound. 
When  fully  exposed,  it  is  well  to  pass  the  sound  under  the 
trachea,  thus  placing  this  on  the  stretch.  The  needle  is 
then  easily  inserted  between  the  cartilaginous  rings.  A 
few  scratches  should  be  made  with  the  needle  on  the  inside 
of  the  trachea,  in  order  to  give  the  organism  a  foot-hold. 
The  amount  of  fluid  injected  should  be  relatively  small. 
Frequently,  the  material  is  introduced  into  the  trachea  by 
means  of  the  platinum  wire.  In  that  case,  one  or  two 
tracheal  rings  are  cut  and  the  mucous  membrane  is  slightly 
injured  by  scratching,  or  by  means  of  a  hot  wire.  The 
wound  is  then  closed,  employing  the  usual  precautions 
.  against  accidental  infection. 

10. — Infection  of  the  alimentary  canal. — A  large  number 
of  diseases  of  man  and  animals  are  due  to  infection  along 
this  tract.  Hence,  in  establishing  the  relation  of  suspected 
organisms  to  such  diseases,  it  is  desirable  to  do  so  by  fol- 
lowing, as  nearly  as  possible,  the  natural  mode  of  infection. 

The  usual  procedure  is  to  administer  the  bacterial  fluid 
with  the  food  or  drink.  When  the  animal  refuses  to  par- 
take it  is  necessary  to  employ  a  stomach-tube.  A  rubber 
tube,  about  five  mm.  in  diameter,  will  answer  this  purpose. 
It  should  be  connected  with  a  small  funnel.  In  order  to 


272  BACTERIOLOGY. 

prevent  -the  animal  from  biting-  the  tube,  this  should  be 
passed  through  a  perforated  piece  of  wood,  or  through  a 
cork-borer  which  is  held  firmly  in  the  mouth.  Any  desired 
amount  of  liquid  can  thus  be  administered  without  difficulty. 

In  experiments  with  the  bacteria  of  cholera  and 
typhoid  fever,  it  has  been  the  practice  to  first  introduce 
into  the  stomach  some  sodium  carbonate  solution.  This 
was  done  in  order  to  neutralize  the  free  hydrochloric  acid 
of  the  stomach,  which  otherwise  might  destroy  the  bacteria 
introduced.  Again,  it  was  customary  to  inject  opium  into 
the  peritoneal  cavity,  in  order  to  retard  or  paralyze  the 
peristaltic  action  of  the  intestines.  The  bacteria  were  thus 
given  every  opportunity  to  develop  in  the  intestinal  canal. 

In  his  classical  studies  on  cholera,  Metschnikov  showed 
that  the  above  precautions  can  be  done  away  with,  and 
better  results  obtained  by  feeding  milk  suspensions  of 
the  germs  to  new-born  rabbits  or  guinea-pigs.  The  intes- 
tines of  the  new-born  animal  are  free,  at  least  for  a  short 
time,  from  bacteria.  The  experimental  organism,  when 
introduced  under  these  conditions,  is  enabled  to  develop 
unhindered.  The  intestinal  flora  of  the  adult  individual 
may,  in  some  cases,  favor  the  growth  of  the  invading  or- 
ganism whereas,  in  other  cases,  it  may  exert  a  positive 
inhibiting  action — microbic  association. 

Occasionally,  it  may  be  desirable  to  avoid  passage  of 
the  material  through  the  stomach.  In  that  case  the  mater- 
ial can  be  injected  directly  into  the  intestines,  but  to  do 
this  properly  necessitates  a  laparotomy.  Such  intra- 
duodenal  injections  are  very  rarely  made. 


Observation  of  Infected  Animals, 

The  inoculated  animal  should  be  placed  by  itself,  in  a 
suitable  cage  or  jar.  Obviously,  where  a  number  of  ani- 
mals, as  guinea-pigs,  are  injected  with  the  same  material, 
they  may  be  placed  in  the  same  cage.  They  should  be 


OBSERVATION  OF  INFECTED  ANIMALS. 


273 


numbered  and  designated  by  their  color,  by  their  curly  or 
smooth  hair,  etc.,  and,  if  need  be,  by  painting  the  nose, 
back  or  flank  with  anilin  dyes.  The  student  should  never 
burden  his  memory  with  details  as  to  dose,  time  of  inocula- 
tion, etc.  A  complete  record  should  be  made  at  the  time 
the  work  is  done,  and  in  this  way  all  uncertainty  is  done 
away  with. 

The  experimental  animals,  as  a  rule,  should  be  weighed 
before  infection,  and  every  day  or  two  afterward.  Each 
weighing  should  be  done,  as  nearly  as  possible,  under  the 
same  conditions  as  the  first  one. 
That  is  to  say,  the  animal  should 
have  plenty  of  jood  and  water  before 
weighing.  In  warm  weather  the  need 
of  water  is  especially  imperative. 
An  experimental  animal  may  show 
a  difference  of  200  or  300  g.  between 
the  two  weighings,  and  this  differ- 
ence may  be  wholly  due  to  the  with- 
holding of  water.  Unless  the  precau- 
tions mentioned  are  observed  the 
weighing  results  will  have  no  value 
whatsoever.  When  properly  carried 
out,  it  affords  the  best  possible  index 

of  the  physical  well-being  of  the  animal.  A  steady,  though 
slight  decrease  in  weight,  is  a  sure  indication  of  a  chronic, 
wasting  disease.  On  the  other  hand,  a  steady  increase  in 
weight  is,  as  a  rule,  a  reliable  index  of  health. 

The  temperature  of  the  animal  should  be  taken  before 
beginning  the  experiment,  and  subsequently  on  each  suc- 
cessive day  at  the  same  hour.  The  ordinary  clinical  ther- 
mometer should  be  inserted  well  up  into  the  rectum.  Pre- 
vious to  insertion,  the  bulb  should  be  covered  with  vaselin. 
The  variations  in  temperature  are  especially  important 
when  studying  the  action  of  bacterial  poisons  on  the  animal 
body. 


FIG.  46.     a— Rat  cage  with 


18 


274 


BACTERIOLOGY. 


The  inoculated  animals  should  be  kept  in  glass  jars,  or 
in  wire  cages  which  can  be  readily  sterilized.  It  is  undesir- 
able and  not  safe  to  keep  such  animals  in  wooden  boxes. 
White  mice  or  rats  can  be  kept  in  battery  jars.  These 
should  be  provided  with  a  wire-gauze  top,  loaded  down  with 
a  mass  of  lead.  Pig.  46  shows  such  a  jar  and  also  the  cru- 
cible or  rat  forceps  ordinarily  used.  Rabbits,  guinea-pigs, 
etc.,  can  be  kept  in  wire  cages  similar  to  the  one  shown  in 
Fig.  47.  By  removing  the  four  thumb-screws  on  the  upper 


FIG.  47.    Vaughan's  cage  for  rabbits,  guinea-pigs,  etc.; 
sterilizable. 

side,  the  ends,  sides  and  top  collapse.  Several  cages  can 
thus  be  arranged  on  top  of  each  other  and  then  placed  in  a 
large,  dry-heat  sterilizer.  The  cage  proper  is  30  cm.  high, 
38  deep  and  54  wide.  The  feet  are  12  cm.  high. 


Post- Mortem  Examination  of  Infected  Animals, 


When  the  infected  animal  dies  it  should  be  examined  as 
soon  as  possible.  After  the  lapse  of  a  few  hours,  especially 
in  warm  weather,  the  body  is  liable  to  become  invaded  by 


POST-MORTEM  EXAMINATION  OP   INFECTED  ANIMALS.  275 

the  common  putrefactive  bacteria  from  the  intestinal  canal. 
Consequently,  in  delayed  examinations,  impure  cultures  are 
apt  to  be  met  with.  Moreover,  an  uncertainty  will  exist 
as  to  whether  the  secondary  invasion  occurred  during*  life, 
or  after  death.  Hence,  in  case  the  animal  cannot  be  exam- 
ined at  once,  it  should  be  placed  in  a  suitable  vessel  and 
kept  on  ice. 


FIG.  48.    a — Searing  iron;  b — Instrument  sterilizing  case. 

All  the  necessary  instruments  should  be  sterilized  be- 
fore use.  This  may  be  done  by  heating  the  instruments,  di- 
rectly in  the  flame.  Inasmuch  as  this  procedure  destroys 
the  temper  it  should  be  avoided  as  much  as  possible.  The 
best  method  of  sterilizing"  instruments,  is  to  place  these  on 
a  perforated  tray  which  is  then  immersed  in  a  copper  or 
enameled  iron  box.  The  box  should  contain  an  almost  sat- 
urated solution  of  borax  (10  per  cent.).  The  borax  is  to  be 
preferred  to  sodium  carbonate,  since  it  prevents  tarn- 
ishing as  well  as  rusting  of  the  instruments.  The  latter 
can  be  kept  constantly  in  the  borax  solution.  When  it  is 
desirable  to  sterilize  these  the  box  is  placed  on  a  Fletcher 
radial  burner.  In  a  few  minutes  the  liquid  will  boil  activ- 
ely. After  3  to  5  minutes  of  boiling,  the  flame  can  be  turned 
off.  This  instrument  sterilizer  is  shown  in  Fig.  48  &. 

A  searing  iron,  also  shown  in  Fig.  48,  several  sterile 
drawn  out  pipettes  (Fig.  61),  and  several  tubes  of  gelatin, 


276  BACTERIOLOGY. 

agar  and  bouillon  should  be  placed  on  the  table.  A  num- 
ber of  clean  cover-glasses  (p.  140),  or  glass  slides  should 
also  be  available. 

The  animal  should  be  nailed  or  tacked  to  a  board.  This  should  be 
about  34  cm.  wide  and  54  cm.  long-.  It  is  well  to  surround  it  with  a 
raised  border,  and  to  fill  any  crack  that  may  be  present  with  melted 
paraffin.  The  board,  thus  prepared,  will  not  leak  and  hence  can  be 
thoroughly  disinfected  after  use.  Instead  of  a  board,  a  galvanized 
iron  or  zinc  tray  can  be  used.  In  the  latter  case  the  animal  is  fast- 
ened by  means  of  strings  tied  to  the  leg's  and  slipped  through  holes 
on  the  side. 

The  animal  is  placed  on  its  back,  on  the  board  or  tray, 
and  its  feet,  extended  as  much  as  possible,  are  fastened 
with  nails  or  cord  as  the  case  may  be.  The  hair  over  the 
abdomen  and  thorax  is  thoroughly  moistened  with  a  cloth 
soaked  in  mercuric  chloride.  With  a  pair  of  sterilized 
forceps,  the  skin  over  the  lower  part  of  the  abdomen  is 
raised  and  a  slight  transverse  nick  is  made  with  sterilized 
scissors.  Into  the  opening  thus  made,  the  lower  blade  of 
the  scissors  is  introduced  and  an  incision  is  made  along  the 
median  line  to  the  neck.  While  making  the  incision,  the 
skin  is  kept  raised  by  means  of  the  forceps  to  avoid  cutting 
through  the  abdominal  or  thoracic  walls.  At  each  end  of 
this  incision,  lateral  cuts  are  made  in  the  direction  of  the 
extremities  and  the  two  flaps  of  skin,  thus  prepared,  are 
carefully  reflected,  thus  exposing  the  entire  abdominal  and 
thoracic  walls.  The  condition  of  the  subcutaneous  tissue, 
of  the  abdominal  walls,  of  the  blood-vessels  and  the 
presence  or  absence  of  pus,  edema,  gas,  etc.,  should  be 
noted. 

Before  proceeding  further,  it  is  well  to  prepare  several 
cover- glasses  from  the  subcutaneous  tissue.  The  cover-glass 
is  placed  on  the  desired  spot.  It  is  then  seized  at  the  edge, 
by  the  forceps,  and  carefully  drawn  off,  not  lifted  up.  If 
much  edematous  fluid  is  present  it  will  be  better  to  take 
up  some  of  this  on  a  looped  wire  and  then  spread  it  over 


POST-MORTEM  EXAMINATION  OF    INFECTED   ANIMALS.  277 

the  cover-glass.  Cultures,  as  a  rule,  are  not  made  from  the 
subcutaneous  tissue  inasmuch  as  they  can  be  obtained  in  a 
more  pure  condition  from  the  peritoneal  cavity,  and  espe- 
cially from  the  heart-blood. 

With  another  sterilized  pair  of  forceps  the  lower  part 
of  the  abdominal  wall  is  raised  and  a  slight  nick  made  with 
sterile  scissors.  While  the  abdominal  wall  is  kept  stretched 
the  lower  blade  of  the  scissors  is  introduced  into  the  cavity 
and  an  incision  is  made  as  far  as  the  diaphragm.  The 
diaphragm  is  then  cut  close  to  the  ribs.  The  costal  carti- 
lages on  both  sides  are  now  cut,  if  need  be,  with  sterile 
bone  forceps.  The  triangular  piece  of  the  thoracic  wall, 
the  sternum  and  ribs,  is  then  wholly  removed.  The  lower 
end  of  the  abdominal  incision  is  extended  toward  the  ex- 
tremities. The  abdominal  walls  can  now  be  turned  back. 

The  entire  abdominal  and  thoracic  cavity  is  thus 
opened  for  inspection.  In  making  the  incisions,  special 
care  must  be  taken  to  avoid  cutting  into  the  intestines,  or 
internal  organs. 

The  condition  of  the  abdominal  and  thoracic  cavities 
should  be  carefully  observed.  The  quantity,  consistence 
and  color  of  the  pleural  or  peritoneal  exudate,  if  any,  is  to 
be  noted.  The  appearance  of  the  peritoneum,  and  the  pos- 
sible presence  of  minute  miliary  tubercles  should  be 0 con- 
sidered. The  color,  size  and  consistency  of  the  liver, 
spleen,  and  kidneys  should  be  observed.  Likewise  the 
lungs,  pericardial  sac  and  the  heart,  whether  it>[is  in  dias- 
tole or  systole,  should  be  examined.  Attention  should  be 
given  to  the  presence  of  abcesses,  tubercles  or  necrotic 
areas. 

The  peritoneal  fluid,  if  it  is  found  on  examination  to  be 
rich  in  bacteria,  can  be  drawn  up  into  a  syringe  and 
injected  into  another  animal.  Or,  the  fluid  may  be  drawn 
up  into  a  sterile,  drawn-out  tube  pipette  (Fig.  61).  This  is 


278  BACTERIOLOGY. 

then  sealed  in  the  flame  and  the  material  can  thus  be  pre- 
served for  future  use.  At  times,  there  is  very  little  exudate 
although  the  bacteria  may  be  present  in  abundance.  In 
that  case,  if  it  is  desired  to  inoculate  direct  into  another 
animal,  some  bouillon  should  be  transferred  by  means  of 
a  drawn-out  pipette  to  the  abdominal  cavity.  By  rubbing 
the  pipette  over  the  surface  of  the  peritoneum  and  drawing1 
up  the  liquid  into  the  pipette,  several  times  in  succession, 
a  good  bacterial  suspension  can  be  obtained.  One  of  the 
best  methods  of  increasing  the  virulence  of  an  organism  is 
by  successive  passage  through  animals. 

With  sterile  forceps  and  scissors,  the  spleen,  liver  and 
kidneys  can  be  transferred  to  sterile  Esmarch  dishes  and 
subsequently  examined.  Pieces  of  these  organs  can  be 
hardened  and  sectioned  (Chapter  XV). 


FIG.  49.    a— The  Roux  spatula  of  nickeled  steel;    b— Nuttall:s  platinum  needle. 

If  it  is  desired  to  make  transplantations  from  any  of 
these  organs,  the  surface  should  first  be  seared  with  a  hot 
knife  or  with  the  searing  iron  (Pig.  48  a).  Portions  of  the 
organ  can  then  be  cut  out  with  sterile  instruments  and 
transferred  to  tubes.  The  sterile  pipette  can  also  be  used 
for  removing  a  portion  of  the  pulp  to  the  culture  media. 
The  spatula  employed  by  Roux  to  remove  diphtheritic 
membranes  is  well  adapted  to  pick  up  pieces  of  tissues. 
These  can  then  be  thoroughly  smeared  over  the  surface  of 
the  medium,  or  squeezed  against  the  walls  of  the  tube. 
Nuttall's  platinum  needle  (Fig.  49  &)  is  likewise  very  use- 
ful in  transferring  bits  of  tissue  to  the  culture  tube. 

The  heart-blood,  as  a  rule,  contains  the  specific  organ- 
ism in  the  purest  condition.  A  large  amount  of  blood  can  be 
drawn  from  the  heart,  especially  if  this  is  done  immediately 


POST-MORTEM  EXAMINATION  OF    INFECTED   ANIMALS.  279 

after  death,  before  coagulation  has  set  in.  This  can  be  easily 
accomplished  in  the  following'  manner:  With  sterile  scissors 
the  pericardial  sac  should  be  opened  and  the  heart  exposed. 
The  apex  of  the  heart  is  grasped  in  a  pair  of  broad  pointed 
forceps,  preferably  arterial  forceps,  and  the  surface  of  the 
heart  is  burned  with  a  red-hot  glass  rod  or  searing-  iron 
(Fig-.  48).  The  point  of  a  sterile,  drawn  out  tube  pipette, 
is  then  broken  or  cut  off.  The  end  of  the  pipette  is  steril- 
ized by  passing-  throug-h  the  name  several  times,  and,  when 
cool,  it  is  passed  through  the  burned  surface  into  the  right 
ventricle.  The  heart  should  be  held  on  a  stretch.  Suction 
is  now  applied  to  the  mouth  of  the  pipette,  and  the  blood 
is  thus  drawn  up  into  the  tube.  The  culture  media  can  be 
inoculated  direct  by  means  of  the  pipette,  or  the  latter  may 
be  sealed  and  the  contents  thus  preserved.  It  may  be  inci- 
dentally remarked  that  the  organisms  thus  kept  in  blood 
preserve  their  vitality  and  virulence  for  a  greater  length  of 
time  than  on  ordinary  media. 

In  many  instances,  it  is  desirable  to  examine  the  mu- 
cous membrane  of  the  stomach  and  intestines  for  hemor- 
rhagic  infiltrations  and  necroses.  The  digestive  tube  in 
that  case  should  be  slit  up  longitudinally  and  washed,  pre- 
ferably in  a  dilute  formaldehyde  or  in  mercuric  chloride 
solution. 

Cover-glass  preparations  should  be  made  from  the 
peritoneal  surface  of  the  abdominal  wall,  or  from  the  sur . 
face  of  the  intestines,  liver,  lungs,  etc.  These  are  easily 
prepared  by  placing  the  clean  cover-glass  on  the  selected 
surface,  and  then  drawing  it  off  by  means  of  a  pair  of  for- 
ceps. A  thin  layer  of  material  adheres  to  the  cover-glass. 

Similar  preparations  should  be  made  from  the  cut  sur- 
faces of  the  various  organs.  For  this  purpose,  the  cover- 
glasses  are  placed  on  a  mounting  board1  and  held  in  place 

lTwo  boards  are  supplied.  The  large  one  is  25  cm.  square;  the 
other  7i  X  20  cm. 


280  BACTERIOLOGY. 

while  the  cut  organ  is  drawn  over  the  surface  of  the  glass. 
The  piece  of  tissue  should  be  held  by  a  pair  of  wide-pointed 
forceps.  Not  infrequently,  the  cut  surface  of  an  organ  is 
very  soft  and  pulpy,  and  if  applied  to  the  cover-glass  will 
leave  a  thick  daub  of  material.  This  can  be  avoided  by 
first  touching  the  tissue  to  a  clean  slide,  or  to  the  bottom 
of  a  Petri  dish.  In  this  way  the  excess  of  fluid  is  removed, 
and  it  becomes  possible  to  spread  a  very  thin,  even  film 
over  each  cover-glass. 

Cover-glass  streaks  should  also  be  made  from  the  heart- 
blood.  A  good  preparation  of  the  blood  should  show  the 
corpuscles  spread  out  in  a  single  layer  and  separated  from 
one  another.  This  cannot  be  done  if  a  platinum  wire  is 
employed  to  spread  the  material.  A  minute  drop  of  the 
blood  should  be  placed  on  the  glass-slip  and  covered  with 
another  one.  The  two  slips  should  then  be  drawn  apart, 
when  a  thin  layer  will  be  left  on  each  cover-glass. 

An  easier  procedure  is  to  place  the  droplet  of  the  blood  on  a 
cover-glass.  A  large  square  cover-glass  (20  mm.)  should  be  cut  into 
halves.  The  edge  of  one  of  these  should  be  touched  to  the  droplet  of 
blood  and  then  drawn  across  the  cover-glass.  The  same  result  can 
be  obtained  with  a  glass-slide.  The  drop  of  blood,  placed  on  one  of 
these,  is  touched  with  the  edge  of  another  slide  which  is  then  drawn 
along  the  surface.  The  slide  is  then  covered  with  alcohol,  for  a  min- 
ute or  two.  This  is  then  washed  away  and  the  stain  applied  for  a  few 
seconds.  The  slide  is  then  washed  and  examined.  For  this  purpose, 
a  cover-glass  can  be  dropped  upon  the  moist  slide.  Or,  the  latter 
may  be  dried  between  filter-papers,  and  a  drop  of  cedar-oil  can  then 
be  placed  directly  on  the  specimen.  If  a  permanent  mount  is  desired 
the  slide  is  freed  of  water  or  oil  by  means  of  paper;  a  drop  of  balsam 
is  then  added  and  covered  with  a  glass-slip. 

The  fixing  of  the  blood  preparations  requires  spe- 
cial care  in  order  to  prevent  alteration  of  the  cor* 
puscles.  A  minimum  of  heat  should  be  used.  The  best 
specimens  are  obtained  by  immersing,  for  a  few  minutes,  in 
absolute  alcohol  or  in  a  mixture  of  equal  parts  of  absolute 
alcohol  and  ether.  When  fixed  in  this  way  the  organisms 


POST-MORTEM  EXAMINATION  OF    INFECTED  ANIMALS.  281 

appear  larger  than  usual,  and  really  beautiful  specimens 
may  be  obtained. 

In  anthrax  and  other  diseases  the  specific  organisms  are  present- 
in  large  numbers  in  the  blood,  and  tend  to  accumulate  in  the  narrow 
capillaries.  Thin  sections  of  the  internal  organs  and  other  tissues, 
on  staining,  will  reveal  the  presence  of  these  bacteria.  Sometimes 
beautiful  demonstrations  of  the  crowding  of  bacteria  into  the  capil- 
laries can  be  obtained  by  making  preparations  of  the  mesentery. 
For  this  purpose  a  large  animal,  rabbit  or  guinea-pig,  is  desirable. 
A  portion  of  the  intestine  should  be  stretched  so  as  to  expose  the 
mesentery.  A  cover-glass  is  dropped  on  the  clean  mesentery,  and 
the  intestine  is  then  laid  over  the  glass-slip.  With  a  pair  of  broad- 
pointed  forceps  the  cover-glass  is  firmly  seized  and  turned  over  so 
that  the  membrane  covers  the  entire  upper  surface.  The  mesentery 
is  then  cut  loose  with  a  pair  of  sharp  scissors.  The  cover-glass  now 
is  covered  on  one  side  by  a  thin  piece  of  the  mesentery.  It  is  placed 
in  an  Esmarch  dish  and  covered  with  absolute  alcohol,  to  which  a  few 
drops  of  formaldehyde  are  added.  The  specimen  is  thus  fixed  in  a  few 
hours,  after  which  it  is  washed  with  water  and  stained  by  Gram's 
method,  if  this  can  be  used.  The  minute  branching  capillaries  will 
often  be  found  plugged  full  of  bacteria.  Appearances  of  this  kind 
gave  rise,  at  one  time,  to  the  hypothesis  that  bacteria  produced  dis- 
ease by  mechanical  interference  with  the  circulation. 

The  cover-glass  preparations  made  from  the  animal 
body,  in  the  manner  indicated,  contain  a  relatively  large 
amount  of  organic  matter.  Consequently,  care  must  be 
taken  to  prevent  over-staining  of  the -back-ground,  inas- 
much as  this  will  interfere  with  a  satisfactory  examination 
of  the  bacteria  that  may  be  present.  The  following  proce- 
dure will  enable  one  to  deeply  stain  the  bacteria,  without 
scarcely  coloring  the  back-ground.  The  importance  of  this 
will  be  recognized  when  but  a  few  organisms  are  present 
on  the  cover-glass. 

The  cover-glass  preparation  is  fixed  with  a  minimum 
amount  of  heat.  The  directions  for  fixing  a  specimen  have 
been  given  on  p.  148. 

When  the  " fixed"  glass-slip  has  cooled,  a  drop  or  two 
of  water  should  be  added  so  as  to  cover  the  material.  A 


282  BACTERIOLOGY. 

drop  or  two  of  the  fairly  strong  solution  of  the  dye  (gentian 
violet,  p.  147),  is  then  added,  and  the  specimen  is  gently 
swayed  for  a  few  seconds  (5-10).  The  cover-glass  is  then 
washed  as  rapidly  as  possible,  and,  after  drying  the  clean 
under  surface,  it  can  be  examined.  Scarcely  any  color  will 
be  seen  by  the  eye,  but  under  the  microscope  the  bacterial 
cells  will  be  seen  deeply  stained,  with  hardly  any  colored 
back-ground.  Hanging-drop  examinations  of  any  exudate 
that  may  be  present,  and  of  the  heart-blood  should  be  made. 

The  necessary  material  for  cultivation  should  be  placed 
in  culture  tubes,  which  can  be  then  set  aside  until  the  micro- 
scopic examination  is  completed  and  the  animal  disposed 
of.  Gelatin  or  agar  plates  are  then  made  from  this  mater- 
ial. The  material  should  also  be  planted  in  bouillon  and 
should  be  streaked  over  the  surface  of  an  inclined  agar 
tube.  Isolated  colonies  may  be  obtained  by  successively 
streaking  a  number  of  agar  tubes  in  the  manner  described 
on  p.  238.  The  agar  and  bouillon  tubes  should  then  be  set 
aside  in  an  incubator  and  allowed  to  develop. 

In  the  post-mortem  work  proper,  the  greatest  care  is 
taken  to  prevent  the  introduction  of  foreign  organisms, 
since  these  might  give  misleading  results.  Throughout  the 
work,  however,  the  operator  must  be  extremely  careful  to. 
prevent  personal  infection.  The  rule  to  sterilize  every  in- 
strument shortly  before  and  immediately  after  use,  before 
it  has  left  the  hands,  must  be  strictly  attended  to.  Direct 
contact  of  the  hands  with  infectious  matter  must  be  care- 
fully avoided,  and  when  such  contact  has  taken  place 
prompt  disinfection  must  be  resorted  to.  It  may  be  well  in 
this  connection  to  call  attention  to  the  precautions  empha- 
sized on  p.  170.  When  blood  or  pieces  of  tissue  adhere  to  the 
instruments,  the  latter  should  not  be  placed  at  once  into  the 
flame,  inasmuch  as  the  sudden  heating  may  cause  the  ma- 
terial to  spurt  and  scatter  about.  To  avoid  this,  the  ma- 


LABORATORY  WORK   WITH  ANTHRAX  ANIMALS.  283 

terial  should  first  be  dried  by  holding  the  instruments  close 
to  the  flame.  This  precaution  should  also  be  observed  when 
sterilizing1  wires  which  are  covered  with  gelatin. 

When  the  examination  is  completed,  the  animal  is 
placed  in  a  jar  and  covered  with  mercuric  chloride  or  other 
disinfectant.  Eventually,  it  should  be  cremated.  If  this 
cannot  be  done,  the  jar  and  contents  should  be  placed  in  a 
sterilizer  and  steamed  for  at  least  one  hour. 

All  instruments  employed  should  be  returned  to  the 
borax  solution  and  boiled  for  about  5  minutes.  The  glass 
pipettes  which  have  been  used,  are  sealed  at  the  lower  end, 
then  strongly  heated  in  a  flame;  after  which,  they  are 
plunged  direct  into  the  water-bath.  The  tube  breaks  into 
fragments,  and  hence  can  now  be  effectively  sterilized  by 
boiling.*  The  nails  should  be  sterilized  in  the  flame  and  the 
board  should  be  treated  with  a  strong  mercuric  chloride 
solution  (1-500). 

Laboratory  Work  with  Anthrax  Animals. 

A  guinea-pig  or  a  rabbit  is  inoculated  subcutaneously 
with  a  small  amount  of  an  agar  growth  of  the  anthrax 
bacillus.  When  the  animal  dies,  in  1^-2  days,  a  post- 
mortem examination  is  at  once  made.  The  material  thus 
obtained  is  used  for  the  following  experiments. 

Isolation  of  the  bacillus  in  pure  culture. — The  bacillus  of 
anthrax  which  is  present  in  the  blood,  tissues  and  organs 
of  the  guinea-pig,  must  be  isolated  and  obtained  in  pure 
culture.  This  can  be  readily  accomplished  by  the  gelatin 
plate  method.  For  this  purpose  a  small  piece  of  liver, 
about  half  the  size  of  a  grain  of  wheat,  is  cut  off  with  a 
pair  of  sterilized  scissors.  The  piece  of  tissue  is  placed  on 
the  loop  of  a  sterilized  platinum  wire  and  transferred  to  a 
tube  of  liquefied  gelatin.  By  rubbing  the  piece,  with  the 
wire,  against  the  walls  of  the  tube  the  blood  can  be 


284  BACTERIOLOGY. 

squeezed  out,  and  the  organisms  present  can  thus  be  spread 
throughout  the  gelatin.  From  this  tube,  which  is  No.  1, 
transfers  are  made  in  the  usual  manner  to  tube  No.  2,  and 
from  this  to  tube  No.  3.  Gelatin  plates  (Petri  dishes)  are 
are'  then  made  in  the  usual  manner  and  set  aside  for  two 
or  three  days  to  develop. 

The  form  of  the  colonies  should  then  be  carefully 
studied,  inasmuch  as  this  is  very  characteristic.  If  possi- 
ble, impression  preparations  of  the  surface  colonies  should 
be  made  and  stained  with  methylene  blue. 

Since  the  colony  is  a  pure  culture  of  the  anthrax  bacil- 
lus, transplantations  to  tubes  in  turn  will  yield  pure  cul- 
tures. A  stab  culture  in  gelatin  and  a  streak  culture  on  in- 
clined agar,  and  on  .two  potatoes  in  tubes,  should  be  made. 
The  inoculation  of  the  inclined  media  is  made  by  simply 
drawing  the  end  of  the  platinum  wire  over  the  middle  of 
the  surface  of  the  agar  or  potato.  The  agar  tube  and 
one  potato  tube  are  placed  in  the  incubator  at  37  to  39°,  for 
one  or  two  days,  then  removed  and  examined  for  threads 
and  spores.  The  other  potato  tube  is  allowed  to  develop 
at  a  temperature  of  15-18°.  Under  these  conditions  spores 
are  not  produced,  but  instead,  marked  involution  forms  will 
be  found. 

A  tube  of  peptonless  agar  should  be  inoculated  at  the 
same  time  as  the  above  and  allowed  to  develop  in  the  incu- 
bator. This  growth,  unlike  that  on  the  ordinary  agar,  will 
be  extremely  rich  in  spores.  The  material  obtained  in  this 
way-  will  be  utilized,  subsequently,  for  the  staining  of 
spores.  The  ordinary  agar  does  not  yield  a  good  supply 
of  spores,  inasmuch  as  the  anthrax  bacillus  employed  has 
been  cultivated  artificially  for  many  years,  and,  as  a  result, 
shows  an  asporogenic  tendency.  If  ^-1  drop  of  calcium 
hydrate  is  added  to  the  tube  of  ordinary  agar,  and  this 
then  sterilized,  it  will  yield  an  abundance  of  spores.  The 
absence  of  pepton  and  the  presence  of  calcium  favor  spore 
formation. 


LABORATORY  WORK  WITH  ANTHRAX  ANIMALS.  285 

Agar  plate  culture. — The  gelatin  plates,  prepared  as 
above,  do  not  always  yield  the  most  characteristic  col- 
onies. Moreover,  owing-  to  the  liquefaction  of  the  gela- 
tin, it  is  difficult  to  obtain  satisfactory  cover-glass  im- 
pressions. The  agar  plates,  on  the  other  hand,  give 
excellent  colonies  from  which  impression  preparations 
can  be  easily  made.  The  method  of  making  agar  plates 
is  as  follows: 

Three  agar  tubes,  each  of  which  contains  about  8  c.c. 
of  agar,  are  selected  and  placed  upright  in  boiling  water. 
When  the  agar  has  become  perfectly  fluid,  the  burner  is 
then  removed  from  under  the  water-bath,  and  the  water 
with  the  immersed  tubes  is  allowed  to  cool  slowly  until  a 
temperature  of  50°  is  reached.  Tube  1  is  then  inoculated 
with  a  piece  of  the  liver,  or  other  organ,  in  the  manner 
described  above  when  making  gelatin  plates.  The  usual 
dilutions  to  tubes  2  and  3,  are  then  made  as  rapidly  as  pos- 
sible. After  withdrawal  of  the  cotton  plug,  the  neck  of 
each  tube  is  flamed  and  the  contents  poured  into  sterile, 
Petri  dishes.  The  plates  are  then  set  aside  in  the  incuba- 
tor at  37°  for  12-18  hours. 

The  nutrient  agar  solidifies  at  about  40°.  Conse- 
quently, rapid  work  is  necessary  in  order  to  inoculate  the 
tubes  and  pour  the  contents  before  solidification  takes 
place.  If  this  does  occur,  the  experiment  must  be  repeated 
with  new  agar  tubes.  Ice- water  must  not  be  used  to  con- 
geal the  agar  plates.  They  will  solidify  without  employ- 
Ing  any  cooling  apparatus. 

When  developed,  the  colonies  should  be  compared  with 
those  on  gelatin;  they  should  be  sketched  and  used  for 
making  impression  preparations. 

Hanging-drop  examination. — Take  a  clean  f  inch  cover- 
glass  and  sterilize  by  passing  it  several  times  through  the 
flame.  Transfer  a  small  drop  of  sterile  bouillon  to  the 
cover-glass  and  then  add  to  it,  with  a  sterilized  wire,  a  min- 


286  BACTERIOLOGY. 

ute  amount  of  the  heart-blood.  Apply  the  concave  slide, 
ringed  with  vaselin,  and  examine  the  hanging-drop  with  the 
No.  7  objective.  Study  the  characteristics  of  the  anthrax 
bacillus  as  it  exists  in  the  blood,  and  compare  its  size 
with  that  of  the  blood-cell.  Then  label  the  slide  and  set 
aside  in  the  incubator  for  24  hours.  Examine  the  same  on 
the  following1  day  and  observe  the  formation  of  threads,  of 
sporogenic  granules  and  possibly  of  spores. 

Owing  to  the  rapid  flow  of  blood,  the  anthrax  bacillus 
cannot  give  rise  to  long  threads  in  the  living  body.  In  a 
quiet  liquid,  as  in  the  drop,  the  tendency  to  form  threads  is 
favored.  Stained,  preparations  can  be  made  from  this  hang- 
ing-drop culture.  The  cover-glass  should  be  moved  to  one 
side,  just  sufficiently  to  permit  the  forceps  to  pick  it  up.  It 
is  then  carefully  raised,  avoiding  contact  with  the  vaselin. 
A  portion  of  the  drop  culture,  transferred  to  a  droplet  of 
water  on  a  clean  slide,  is  spread  out,  dried,  fixed  and  stained 
in  the  usual  way. 

Stained  preparations. — Place  about  18  clean  cover-glasses 
on  the  small  mounting  board  (7-J-  X  20  cm.),  or  on  the  lid  of 
a  slide-box.  Pick  up  a  piece  of  the  spleen,  kidney  or  liver 
in  a  pair  of  forceps,  and,  while  holding  the  cover-glass  down 
with  another  pair,  lightly  pass  the  cut  surface  of  the 
organ  over  the  cover-glass.  A  very  thin  and  even  film 
is  desirable  (see  p.  280).  In  this  way  streak  all  the  cover- 
glasses;  some  with  spleen,  others  with  liver  and  the  remain- 
der with  kidney  tissue.  Keep  each  set  distinct.  Then  al- 
low the  specimens  to  dry  in  the  air  and  fix  cautiously  by 
passing  once  through  the  flame.  Special  care  must  be  taken 
not  to  over-heat,  inasmuch  as  the  anthrax  bacillus  in 
that  case  will  not  stain  readily.  If,  on  subsequent  trial, 
a  cover-glass  is  found  to  be  fixed  imperfectly  the  others 
can  be  given  an  additional  passage  through  the  flame. 
These  cover-glasses  are  commonly  known  as  streak  prepar- 
ations. 


LABORATORY  WORK   WITH  ANTHRAX  ANIMALS.  287 

Stain  some  of  the  fixed  cover-glasses  with  simple  anilin 
•dyes,  such  as  gentian  violet  or  fuchsin,  observing  the  sug- 
gestions made  on  p.  281.  Examine  and  study  the  specimen^ 
carefully  and  make  permanent  preparations.  Note  the  or- 
gan in  which  the  bacilli  are  most  abundant.  The  remain- 
der of  the  cover-glasses  will  serve  for  double-staining  by 
Gram's  method. 

Stained  preparations  of  the  heart-blood  (p.  280),  should 
also  be  made. 

Gram's  method. — This  excellent  method  of  demonstrating 
the  presence  of  certain  bacteria  in  the  fluids  and  tissues  of 
the  body  is  based  upon  the  fact  that  the  protoplasm  of  the 
bacterial  cell,  when  stained  with  anilin-water  gentian  violet 
and  then  treated  with  iodine,  forms  a  difficultly  soluble  com- 
pound. By  proper  exposure  to  a  solvent  the  dye  can  now 
be  removed  from  the  entire  cover-glass,  but  not  from  the 
bacterial  cell.  The  deeply  stained  violet  bacteria  lie  on  a 
colorless  back-ground  which  on  treatment  with  a  contrast 
color,  as  eosin  or  picro-carmin,  becomes  stained  a  light 
pink.  The  method  is  as  follows: 

A  solution  of  anilin-iuater  gentian  violet  is  first  prepared. 
Anilin  oil  is  placed  in  a  test-tube  to  a  depth  of  about  half 
an  inch.  The  tube  is  then  filled  with  water,  closed  with  the 
thumb,  and  thoroughly  shaken  in  order  to  obtain  a  satur- 
ated aqueous  solution  of  anilin.  The  liquid  is  then  passed 
through  a  small  filter  and  collected  in  another  test-tube. 
The  filtrate  should  be  perfectly  clear,  not  cloudy.  To  the 
anilin  water  thus  obtained  a  saturated  alcoholic  solution  of 
gentian  violet  is  added  till  the  fluid  is  deeply  colored,  and 
opaque.  This  result  is  obtained  when  0.3 — 0.5  c.c.  of  the 
saturated  gentian  violet  is  added  to  10  c.c.  of  the  anilin- 
water. 

Some  of  the  anilin-water  gentian  violet  thus  prepared 
is  poured  out  into  a  watch-glass,  or  better,  into  a  wide  Es- 
march  dish.  The  fixed  cover-glass  is  placed  between  the 


288  BACTERIOLOGY. 

thumb  and  forefinger,  with  the  specimen  side  down,  and 
then  carefully  dropped  upon  the  surface  of  the  stain.  It  is 
allowed  to  float  on  the  dye  for  3-5  minutes.  Sometimes,  it 
is  necessary  to  stain  for  a  longer  period,  or  to  warm  the 
dye  on  the  radiator  or  on  an  iron  plate  (Pig.  22,  p.  150), 
in  order  to  obtain  a  rapid  and  intense  stain.  The  best 
results  are  obtained  when  the  specimen  is  treated  with  a 
strong  dye  for  a  few  minutes.  The  longer  the  dye  acts, 
the  more  difficult  it  will  be  to  subsequently  obtain  a  good 
decoloration. 

The  cover-glass  is  then  picked  up  with  the  forceps, 
thoroughly  washed  with  water,  and  immersed  in  a  solution 
of  iodine  in  potassium  iodide.  This  is  made  by  dissolving  2 
g.  of  potassium  iodide  and  1  g.  of  iodine  in  300  c.c.  of  dis- 
tilled water.  The  specimen  is  allowed  to  remain  in  the 
iodine  solution  for  3  to  5  minutes.  Care  must  be  taken  not 
to  expose  the  specimen  too  long  to  the  action  of  iodine,  in- 
asmuch as  this  tends  to  cause  the  protoplasm  to  contract 
into  granules.  The  cover-glass  is  then  removed  from  the 
iodine  solution,  washed  with  water,  and  placed  in  95  per 
cent,  or  in  absolute  alcohol.  This  can  be  kept  in  a  watch- 
glass  or  in  an  Esmarch  dish,  which  should  be  tilted  from 
time  to  time  to  facilitate  the  decoloration.  In  the  case  of 
greatly  over-stained  specimens,  a  drop  of  dilute  acetic  acid 
may  be  added  to  the  alcohol. 

From  time  to  time,  the  cover-glass  should  be  washed 
with  water  and  examined  with  the  No.  7  objective  in  order 
to  ascertain  the  progress  in  decoloration.  If  the  material 
is  spread  over  the  cover-glass  in  a  perfectly  thin,  even 
layer  the  decoloration  will  be  rapid  and  thorough.  On  the 
other  hand,  if  thick  masses  are  present,  it  will  not  be  pos- 
sible to  obtain  complete  decoloration  without  also  decolor- 
ing many  of  the  bacteria.  When,  therefore,  the  greater 
part  of  the  back-ground  has  been  decolored,  the  treatment 
with  alcohol  should  be  discontinued. 

The  cover-glass  is  then  washed  with  water  and  stained 


LABORATORY  WORK   WITH   ANTHRAX   ANIMALS.  289 

with  dilute  eosin  for  i  to  i  minute.  The  eosin  is  an  acid 
anilin  dye  and  therefore  stains  the  protoplasm  of  cells, 
nuclei,  etc.,  but  not  bacteria  (p.  146).  Care  must  be  taken_ 
not  to  overstain  the  preparation  with  eosin,  as  it  would 
tend  to  diminish  the  sharp  contrast  that  is  desired.  More- 
over, the  eosin  is  liable  to  decolor  the  violet  bacilli.  After 
staining1  with  eosin,  the  cover-glass  is  thoroughly  washed 
with  water  and  examined  under  the  microscope.  It  should 
show  the  deeply  stained  violet  bacilli  on  a  light  pink  back- 
ground. If  the  specimen  is  satisfactory  it  can  then  be 
floated  off  the  slide,  dried  in  the  air  and  finally  mounted  in 
balsam. 

Weigert's  picro-carmin  solution,  or  Bismarck  brown 
can  also  be  used  for  contrast  colors.  The  Gram-Weigert's 
fibrin  stain,  as  given  in  Chapter  XV,  can  be  used  to  advan- 
tage where  sometimes  the  ordinary  Gram's  method  fails. 
As  will  be  seen,  many  important  organisms  are  not  stained 
by  this  method. 

The  gentian  violet  employed  in  Gram's  method  may  be 
substituted  by  other  para-rosanilin  dyes,  such  as  methyl 
violet,  or  Victoria  blue.  The  rosanilin  dyes,  such  as 
fuchsin,  methylene  blue,  and  vesuvin  will  not  react  with 
iodine. 

Gram's  method  is  applicable  to  many  pathogenic  bacilli 
and  to  most  of  the  micrococci.  A  notable  exception  among 
the  latter  is  the  gonococcus. 

The  following  organisms  are  stained  by  Gram's  method: 

B.  anthracis.  B.  tetani. 

B.  anthracis  symptomatici  (p.  298).  B.  tuberculosis. 

B.  diphtheriae.  M.  pneumonias  crouposae. 

B.  leprae.  M.  tetragenus. 

B.  murisepticus.  Moulds. 

B.  oedematis  maligni  (p.  300).  Staphylococci. 

B.  oedematis  maligni,  No.  II.  Streptococci. 

B.  rhusiopathiae  suis.  Streptothrix  actinomyces. 

Yeasts. 

19 


290  BACTERIOLOGY. 

The  following'  are  not  stained  by  Gram's  method: 

B.  anthracis  symptomatici  (p.  298).    B.  pneumonias. 

B.  cholerae  g-allinarum.  B.  rhinoscleromatis. 

B.  choleras  suis.  B.  typhosus. 

B.  coli  communis.  M.  gonorrheas. 

B.  icteroides.  Spirillum  Obermeieri. 

B.  influenzas.  Vibrio  cholerae  Asiaticas. 

B.  mallei.  V.  Deneke. 

B.  oedematis  maligni  (p.  300).  V.  Finkler-Prior. 

B.  pestis  bubonicae.  V.  Metschnikovi. 

The  following1   synopsis  of  the   staining  methods   for 
streak  preparations  will  be  of  service: 

Cover  glass  preparation. 

Dry  in  air. 

Once  through  flame. 

Simple  stain:  Gram's  stain: 

Add  drop  of  water.  Anilin-water  gentian  violet  (3  to 

Add  dilute  dye  (#-^  min.).  5  min.;  if  hot,  1  to  3  min.). 

Wash  in  water.  Wash  in  water. 

Examine  in  water.  Iodine  in  potassium  iodide  (3  to 

Dry  in  air.  5  min.). 

Mount  in  balsam.  Wash  in  water. 

Decolor  in  absolute  alcohol. 

Wash  and  examine  in  water. 

Contrast  color(dil.eosin,few  sec.). 

Wash  and  examine  in  water. 

Dry  in  air. 

Mount  in  balsam. 

The  above  examinations  of  hanging-drop  and  stained 
preparations  serve  the  purpose  of  demonstrating  the  pres- 
ence of  the  anthrax  bacillus  in  the  different  organs  and 
tissues  of  the  body.  The  form,  size,  etc.,  of  the  bacillus 
found  under  these  conditions  should  be  compared  with  the 
characteristics  of  the  organism  when  grown  in  pure  culture 
on  the  different  media.  For  this  purpose  make  hanging  - 
drop  examinations  and  permanent  simple  stains  of  the 


LABORATORY  WORK  WITH  ANTHRAX  ANIMALS.  291 

bacillus  grown  in  stab  culture  in  gelatin,  in  bouillon,  on 
ordinary  agar,  and  on  peptonless  agar.  The  preparation 
of  impression  cover-glasses  of  the  anthrax  colonies  and 
simple  stains  of  the  bouillon  hanging-drop  culture  have  been 
mentioned. 

Double  staining  of  spores.  — The  growth  of  the  anthrax 
bacillus  on  peptonless  agar,  or  on  potato  at  37°,  when  exam- 
ined in  the  hanging-drop,  will  show  the  presence  of  an 
abundance  of  bright,  highly  refracting  oval  bodies  or 
spores.  These  may  be  observed  free  and  also  within  the 
parent  cell.  Simple  stains  of  this  material  with  fuchsin, 
etc.,  will  show  the  bacilli  deeply  stained,  whereas  the 
spores  remain  colorless.  This  is  undoubtedly  due  to  a 
special  composition  of  the  spore  contents,  as  well  as  to  the 
dense  impenetrable  wall  which  surrounds  the  spore  and 
prevents  the  dye  from  passing  into  the  interior  (p.  55).  By 
proper  treatment  with  strong  anilin  dyes  it  is  possible  to 
force  the  stain  into  the  spore.  Once  within  the  spore,  it  is 
as  -difficult  to  remove  the  dye  as  it  was  to  cause  it  to  enter. 
By  suitable  decoloration  it  is,  therefore,  possible  to  remove 
the  stain  from  everything  on  the  cover-glass,  except  from 
the  spores.  Then,  on  the  application  of  a  contrast  color 
the  specimen  will  show  a  bright  red  spore  within  a  blue  bac- 
illus. The  method  of  double  staining  spores  is  as  follows: 

The  cover-glass  preparation  from  the  peptonless  agar 
is  dried  in  the  air  and  fixed  in  the  usual  manner.  The 
cover-glass  is  held  in  the  forceps  in  the  left  hand,  with  the 
specimen  side  up,  and  covered  with  a  solution  of  hot  car- 
bolic fuchsin.  This  is  held  over  a  Bunsen  flame,  so  that 
vapors  are  given  off  from  the  liquid.  Active  ebullition 
should  be  avoided.  -  From  time  to  time  the  liquid  which  is 
lost  by  evaporation  is  replaced  by  a  fresh  addition  of  the 
carbolic-fuchsin,  and  under  no  condition  should  the  dye  be 
allowed  to  dry  down  on  the  cover-glass.  The  best  results  in 
heating  are  obtained  when  the  flame  is  turned  low,  so  that 


292  BACTERIOLOGY. 

it  is  not  over  two  inches  high.  After  heating"  the  specimen 
in  this  manner  for  two  or  three  minutes,  the  stain  is  tho- 
roughly washed  off  with  water  and  the  cover- glass  exam- 
ined with  the  No.  7  objective.  Colorless  spores  should  no 
longer  be  visible,  but  everything  should  be  stained  a  deep 
red.  If  the  spores  are  not  colored  the  heating  with  car- 
bolic fuchsin  is  repeated  until  they  become  stained. 

The  cover-glass  may  be  floated  on  hot  carbolic  fuchsin  in  an  Es- 
march  dish  for  ^  to  1  hour.  The  carbolic  fuchsin,  whether  in  the 
bottle  or  in  the  Esmarch  dish,  can  be  heated  on  the  iron-plate  as 
shown  in  Fig.  22,  p.  150.  Anilin-water  fuchsin  can  be  prepared  in  the 
same  way  as  the  stain  which  is  employed  in  Gram's  method  (p  287), 
and  can  be  used  in  place  of  carbolic  fuchsin.  It  will  give  excellent 
results. 

The  cover-glass  with  the  deeply  stained  spores  is  then 
placed  in  dilute  alcohol  and  gently  moved  about.  From 
time  to  time,  it  should  be  washed  with  water  and  examined 
with  the  No.  7  objective.  As  soon  as  the  bacilli  are  de- 
colored, the  washing  in  alcohol  is  discontinued.  The  spec- 
imen then  shows  bright  red  spores  within  cells  that  are 
almost  or  wholly  colorless.  The  cover-glass  is  then  stained 
for  a  short  time  with  methylene  blue,  washed  with  water 
and  examined.  The  spores  should  be  stained  deep  red, 
while  the  bacillus  itself  should  be  light  blue. 

Spores  may  be  readily  simple  stained  by  passing  the 
cover-glass,  after  it  has  been  fixed,  8  or  10  times  through 
the  flame.  The  specimen  is  then  heated  for  one  to  two  min- 
utes with  carbolic  fuchsin,  washed  with  water  and  exam- 
ined. The  spores  are  deeply  stained,  but  the  cell  proper  is 
not.  This  excessive  heating  disintegrates  or  weakens  the 
spore-wall,  and  thus  the  dye  is  enabled  to  enter.  Unfor- 
tunately, the  cell-wall  proper  is  destroyed  and  hence  will 
not  stain. 

The  carbolic-fuchsin  (ZiehVs  solution),  is  prepared  by 
adding  1  g.  of  fuchsin  and  13  c.c.  of  absolute  alcohol  to  100 
c.c.  of  5  per  cent,  carbolic  acid.  The  liquid  is  heated  on 


LABORATORY  WORK  WITH  ANTHRAX  ANIMALS.  293 

the  water-bath  until  everything-  dissolves,  and  the  solution 
has  a  clear,  deep  bright-red  color. 

The  carbolic  fuchsin  prepared  as  above  will  keep  for  a  consid-" 
erable  length  of  time.  It  should  always  be  warmed  on  the  iron-plate 
in  order  to  bring-  the  constituents  into  solution.  Eventually,  an 
insoluble  deposit  forms  and  the  solution  becomes  more  transparent, 
weaker,  and  hence  stains  less  energetically  than  in  the  beginning-. 
Instead  of  preparing-  a  larg-e  amount  of  carbolic  fuchsin  it  is  better 
to  dissolve  the  constituents  separately  and  to  bring-  these  together  in 
small  quantity,  whenever  desired. 

For  this  purpose  8  g.  of  fuchsin  are  dissolved  by  heat  in  120  c.c.  of 
absolute  alcohol.  A  deposit  may  form  on  cooling-,  hence  before  meas- 
uring- out  the  liquid  it  should  be  warmed  till  complete  solution  results. 
15  c.c.  of  this  solution  contain  1  g-.  of  fuchsin.  The  addition  of  7.5 
c.c.  of  the  liquid  to  50  c.c.  of  5  per  cent,  carbolic  acid  will,  on  warm- 
ing-, give  an  excellent  Ziehl  solution.  Tubercle  bacilli  which  will  not 
stain  with  old  carbolic  fuchsin  will  promptly  react  with  a  fresh  solu- 
tion prepared  in  the  manner  described. 

The  following  summary  will  be  of  assistance  when 
staining  spores: 

Cover-glass  preparation. 
Dry  in  air. 


Simple:  Double: 

Pass  12  times  or  less  throug-h         Pass  once  throug-h  flame. 

flame.  Carbolic  fuchsin  (hot  2  to  5  min.). 

Carbolic  fuchsin  (hot  i  min.).  Wash  and  examine  in  water. 

Wash  in  water.  Wash  in  dilute  alcohol. 

Examine  in  water.  Wash  and  examine  in  water. 

Dry  in  air.  Contrast  color  (methylene  blue, 

Mount  in  balsam.  i  -i  min.). 

Wash  and  examine  in  water. 

Dry  in  air. 

Mount  in  balsam. 

Phagocytes. — According  to  Metchnikoif' s  cellular  theory 
of  immunity,  the  white  blood  cell  is  endowed  with  the 
power  of  taking  into  itself,  and  ultimately  destroying,  the 
invading  organism.  Phagocytic  action  can  be  readily 
demonstrated  in  frogs  inoculated  with  anthrax.  For  this 


294  BACTERIOLOGY. 

purpose  a  pure  culture  of  the  anthrax  bacillus  is  injected 
into  the  dorsal  lymph  sac  of  a  frog,  and  at  the  of  12  or  18 
hours  the  animal  is  killed  with  chloroform. 

Cover-glass  preparations  should  be  made  with  the  fluid 
in  the  dorsal  lymph  sac  and  stained,  some  with  simple 
anilin  dyes  and  others  by  Gram's  method. 

Animal  inoculations. — A  pure  culture  of  the  anthrax 
bacillus,  isolated  from  the  dead  animal,  can  be  used  to  in- 
oculate a  white  mouse,  a  white  rat  or  a  rabbit.  In  the 
animal  thus  inoculated  the  organism  can  in  turn  be  detected 
and  isolated.  The  rules  of  Koch  can  be  easily  demonstrated 
with  reference  to  the  anthrax  bacillus,  thus  showing,  conclu- 
sively that  it  is  the  cause  of  the  disease. 

Summary  of  Laboratory    Work    with  Anthrax. 


From  the  guinea-pig-  make  : 
Gelatin  plates. 

Colonies. — Impression  preparations. 
Stab  culture  in  gelatin. 

Agar  streak  culture. 

.  .  Examine  each  of  these 

Peptonless  agar  streak. 

^   .  pin  the  hanging-drop,  and 

Potato  tube  culture. 

_   ,  make  permanent  mounts. 

Potato  tube  at  15°. 

Bouillon  tube  culture. 

Agar  plates  at  37°.— Impression  preparations. 
Hanging-drop  culture  of  heart-blood. — Permanent  mounts. 
Streak  preparations  of  blood,  spleen,  liver,  kidney,  lung,  stained 

by  simple  and  by  Gram's  method. 
Inoculate  a  frog  with  a  pure  culture  of   anthrax  and   examine  the 

lymph  fluid  by  the  simple  and  by  Gram's  method  for  phagocytes. 
Simple  and  double  staining  of   spores,  developed  on  peptonless  agar 

and  on  potato  at  37°. 
Involution  forms  on  potato  at  15°. 
Sketch  characteristic  growths  and  forms. 

Inoculation  of  other  susceptible  animals,  thus  proving  that  this  organ- 
ism is  the  cause  of  a  disease. 


CHAPTER   XI. 
THE  PATHOGENIC  BACTERIA. 


B.    ANTHRACIS.— B.    ANTHRACIS   SYMPTOM ATICI.—  B.    CEDEMATIS   MALI- 
GNI. — B.  CEDEMATIS  MALIGNI  NO.  II. — B.  TETANI. — B.  TUBERCULOSIS. 
— B.     LEPR^E.— B.     MALLEI.— B.     DIPHTHERIA.—  M.     PNEUMONLE 
CROUPOS^S.— B.  PNEUMONIA.— B.  RHINOSCLEROMATIS.  — VlBRIO 
CHOLERA  ASIATIC^.— VIBRIO   DENEKE.  —  VlBRIO    FINKLER- 
PRIOR. — VlBRIO  METSCHNIKOVI. — B.    COLI   COMMUNIS. — B. 
TYPHOSUS.— B.     ICTEROIDES.— B.    PESTIS    BUBONICLE.— 
B.      INFLUENZA.—  B.      PYOCYANEUS.—  STREPTOCOC- 
CUS   PYOGENES.  —  STAPHYLOCOCCUS    PYOGENES 

AUREUS-  —  MlCROCOCCUS       GONORRHEA.  —  M. 

TETRAGENUS.    —    SPIRILLUM        OBERMEIERI. 

—     B.       CHOLERA       GALLINARUM.     —     B. 

CHOLERA        SUIS.     -         B.         RHUSIOPA- 

THLE     SUIS.    —    B.        MURISEPTICUS- 


295 


Bacillus  Anthracis,  Davaine,  Pollender  (1849). 

ANTHRAX,      SPLENIC      FEVER     (in     cattle):      WOOL-SORTER'S     DISEASE, 

MALIGNANT  PUSTULE  (in  man);  MILZBRAND  (Germ.)', 

CH ARSON,  SANG  DE  RATE  (-FV.). 

ORIGIN. — Found  in  the  blood  and  tissues  in  anthrax. 

FORM. — Large,  clear,  homogeneous  rods,  with  slightly  rounded 
ends;  size  varies  with  different  media,  but  the  length  is  less  than  the 
diameter  of  a  blood  cell.  Occurs  in  blood  in  short  threads  of  2-4-6 
cells,  which  may  show  slightly  swollen  ends.  In  bouillon  and  on  agar 
it  forms  long  threads.  Involution  forms  develop  on  potato  at  16°. 

MOTILITY.— It  has  no  motion. 

SPORULATION. — Forms  median,  oval  spores,  without  enlargement 
of  cell.  After  long  cultivation  it  may  lose  the  property  of  forming 
spores — asporogenic  variety.  In  such  cases,  growth  on  peptonless 
agar,  or  the  addition  of  i-l  drop  of  Ca(OH)2  to  an  agar  tube,  favors 
spore  formation.  Optimum  temperature,  30°.  Not  formed  below  16° 
or  above  42°,  or  within  the  body.  Spores  possess  variable  resistance. 

ANILIN  DYES. — It  stains  readily,  also  by  Gram's  method. 

GROWTH.  —Is  rapid. 

Gelatin  plates. — Deep  colonies  form  round,  granular,  yellowish-brown  masses,  with 
irregular  borders.  Surface  colonies  are  very  characteristic,  and  according  to  the  consis- 
tency of  the  gelatin  the  border  is  fibrillated  or  shows  very  wavy  strands  of  threads — Medusa 
head.  This  typical  colony  is  readily  obtained  on  agar  plates  grown  at  37°.  Liquefaction. 

Stab  culture. — Short  threads  radiate  from  the  line  of  inoculation  into  the  surrounding 
gelatin,  imparting  a  brush-like  appearance.  Cup-shaped  liquefaction  forms  on  top  and 
gradually  extends  till  the  contents  are  wholly  liquefied.  The  mass  of  bacteria  settles  to 
the  bottom  and  leaves  a  perfectly  clear  solution  above,  without  scum. 

Streak  culture.— On  agar,  it  forms  a  dry,  grayish-white,  slightly  folded  growth.  On 
Potato,  the  growth  is  abundant,  white,  cream-like  and  rather  dry,  and  is  rich  in  spores  (37°); 
or  in  involution  forms  (16°). 

In  bouillon,  a  thick  gelatin  ring  forms  on  the  surface.  Milk  is  coagulated,  the  cas- 
ein is  then  peptonized.  Acid  production  on  lactose  media. 

OXYGEN  REQUIREMENTS,— It  is  aerobic,  but  can  grow  in  the  body  as 
a  facultative  anaerobe. 

TEMPERATURE. — Growth   at  12 — 45°.      The  optimum  is  about  37°. 

BEHAVIOR  TO  GELATIN.  -Liquefies  slowly. 

ATTENUATION.— By  heating  for  10  minutes  at  55°;  i-1  minute  at 
100°.  By  growing  at  42-5°  for  four  weeks.  By  action  of  chemicals  as 
mercuric  chloride,  carbolic  acid,  etc.  By  insolation.  By  growth  un- 
der pressure.  In  the  body  of  immune  animals,  as  frogs. 

IMMUNITY. — Obtained  with  attenuated  cultures,  1st  and  2nd  vac- 
cine of  Pasteur;  with  sterilized  cultures;  with  purified  toxin.  Blood 
of  anthrax  animals  heated  to  55°  protects.  The  blood-serum  of  an  ani- 
mal vaccinated  against  the  bacillus  is  preventive,  and  to  some  extent 
curative. 

PATHOGENESIS. — White  mice,  guinea-pigs,  rabbits,  sheep,  cattle, 
horses  and  man  are  susceptible.  Dogs,  old  white  rats,  cats,  Algerian 
sheep,  birds  and  frogs  are  insusceptible.  Young  animals,  as  dog  or 
rat,  are  more  susceptible  than  old  ones.  Subcutaneous  application 
kills  guinea-pigs  in  24-48  hours.  Post-mortem  examination  shows  sub- 
cutaneous edema  and  enlarged  spleen.  Bacilli  everywhere;  leave 
body  in  bloody  discharges  from  the  nose,  intestine,  urine,  etc. 

INFECTION. — (1)  Through  the  food,  presence  of  spores. — Intestinal 
anthrax  in  sheep  and  cattle.  (2)  Through  wounds,— Inoculation  an- 
thrax in  man  (malignant  pustule).  (3)  Through  the  air, — Lung  an- 
thrax in  man,  the  wool-sorter's  and  possibly  rag-picker's  disease. 

DIAGNOSIS.  -  Microscopical  examination  of  spleen,  blood,  or  of  con- 
tents of  pustule;  isolation  of  pure  culture.  Characteristics  of  the  lat- 
ter and  effect  on  white  mouse  or  guinea-pig.  It  does  not  appear  in 
the  blood  until  shortly  before  death.  It  is  present  in  the  "  rusty  "  or 
dark-colored  sputum  of  the  "wool-sorter's  disease." 

296 


DRAWINGS.  297 


Bacillus  Anthracis  Symptomatic!,  Feser  &  Bellinger  (1878). 

SYMPTOMATIC  ANTHRAX,  BLACK  LEG,  QUARTER  EVIL;   CHARBON  SYMP- 
TOM ATIQUE  (Fr.)',  RAUSCHBRAND  (Germ.). 

ORIGIN.— In  the  subcutaneous  tissue,  muscles,  serous  exudate, 
etc.,  of  symptomatic  anthrax. 

FORM. — Rather  large,  narrow  rods,  with  distinctly  rounded  ends; 
almost  invariably  single,  may  form  in  pairs.  About  three  times  as- 
long  as  wide.  Involution  forms  appear  in  old  cultures — swollen  in  the 
middle  or  at  the  ends. 

MOTILITY. — Actively  motile.  Spore-bearing  rods  eventually  lose 
their  motion.  Shows  lateral  flagella:  giant  whips  are  common. 

SPORULATION. — Spores  develop  readily  as  bright  oval  bodies,  near 
one  end  which  is  enlarged.  Not  formed  in  body  till  after  death. 

ANILIN  DYES.— Stain  readily.  Gram's  method  is  applicable  if  a 
strong  dye  acts  for  some  time.  The  spores  are  readily  double  stained. 

GROWTH.— Rapid,  and  gives  off  a  strong  butyric  acid  odor.  Acid 
or  alkaline  glucose  media  are  best.  Requires  anaerobic  conditions. 

Plates.— On  gelatin,  forms  irregular  masses  surrounded  by  a  dense  whorl  of  threads. 
Liquefies.  On  agar,  the  colonies  vary.  Usually  appears  as  a  dense  mass  of  threads. 

Stab  culture. — In  glucose  gelatin  development  takes  place  in  the  lower  part  of  the 
tube;  the  contents  are  liquefied  and  gas  is  produced.  Energetic  growth  and  gas  produc- 
tion in  glucose  agar.  The  contents  of  the  tube  are  torn  into  several  parts.  Giant  whip& 
common  (Novy). 

Streak  culture. — On  glucose  agar,  in  hydrogen,  a  whitish  spreading  film  forms.  On 
blood  serum  good  growth;  giant  whips  (Loffler). 

Bouillon.— Becomes  cloudy;  gas  bubbles  accumulate  on  the  surface;  after  several 
days  the  growth  settles  to  the  bottom,  forming  a  compact,  adherent  sediment.  Liquid 
above  remains  cloudy  for  several  days. 

Glucose  gelatin  colored  with  litmus. — Develops  growth  in  incubator  under  ordinary 
conditions.  The  color  of  the  litmus  disappears  (reduction),  then  changes  to  a  wine-red, 
showing  formation  of  acids.  Heavy  flocculent  sediment  on  the  bottom. 

Milk.— The  casein  is  quickly  coagulated.    Starch  is  not  inverted.    Grows  on  potato. 

OXYGEN  REQUIREMENTS. — Is  an  obliyative  anaerobe.  Grows  in  vacuum^ 
hydrogen,  carbonic  acid,  etc.  Also  in  glucose  litmus  gelatin  in  air. 

TEMPERATURE.  —  Grows  slowly  at  room  temperature.  Best  at  37-38° . 

BEHAVIOR  TO  GELATIN. — Liquefies. 

AEROGENESIS. — Energetic  production  of  gas,  having  a  disagree- 
able odor;  is  inflammable  and  consists  of  marsh-gas,  hydrogen,  etc. 

ATTENUATION. — Bouillon  cultures  soon  lose  virulence,  but  main- 
tain their  vitality.  Attenuation  takes  place  at  42-43°.  Dry  spore 
bearing  material  heated  to  80°  or  100°  becomes  attenuated.  Viru- 
lence restored  by  inoculating  animals,  and  at  the  same  time  injecting 
some  lactic  acid.  Virulence  maintained  in  solid  media. 

IMMUNITY.— Obtained  (1)  by  inoculating  small  amounts  of  virulent 
germ;  (2)  by  intravenous  injections;  (3)  by  injecting  heated  cultures. 
100°  or  80°;  (4)  inactive  old  cultures;  (5)  filtered  cultures. 

PATHOGENESIS.— Young  cattle,  sheep,  goats,  guinea-pigs,  mice,  are 
highly  susceptible.  Horse,  ass,  white  rat  are  less  so;  while  hogs, 
dogs,  cats,  ordinary  rats,  rabbits,  doves,  ducks,  chickens,  are  almost 
immune.  Subcutaneous  injection  in  guinea-pigs  produces  death  in 
24-48  hours.  An  extensive  subcutaneous  bloody  edema  is  present.  The 
muscles  are  dark,  infiltrated,  and  gas  is  present. 

INFECTION.— Takes  place  naturally  by  inoculation  through  deep 
wounds;  very  rarely  through  the  food.  Poisoned  arrows  used  in  fish- 
ing in  Norway. 

DIAGNOSIS.— Especially  a  disease  of  young  cattle,  not  of  man. 
Difficult  to  distinguish  from  bacillus  of  malignant  edema.  Inocula- 
tion of  the  rabbit  negative;  absence  of  threads;  tendency  to  involu- 
tions. Distinguished  from  anthrax  bacillus  by  form,  motility,  posi- 
tion of  spores,  cultural  properties,  and  by  its  distribution  in  the  body. 

298 


DRAWINGS.  299 


Bacillus  CEdematis  Maligni,  Pasteur  (1877). 

VIBRION    SEPTIQUE     (of     Pasteur).      MALIGNANT     EDEMA;     SEPTICEMIE 
GANGRENE  GAZEUSE  (Fr.)'<   MALIGNES  CEDEM  (Germ). 

ORIGIN. — From  animals  inoculated  with  garden  soil;  from  in- 
fected horse  and  from  man.  In  putrid  liquids,  and  in  intestines. 

FORM. — Rods  about  three  times  as  long-  as  wide,  with  rounded 
ends;  usually  single,  but  may  form  threads  especially  in  the  body. 
In  size,  etc.,  resembles  the  bacillus  of  S.  anthrax;  is  narrower  than 
the  anthrax  bacillus. 

MOTILITY. — Actively  motile.  Lateral  flagella;  giant  whips  (Novy). 

SPORULATION. — In  bouillon  and  agar,  spores  appear  in  24  hours. 
The  best  temperature  is  about  37°.  The  spores  are  median  or  nearly 
so,  with  corresponding  enlargement  of  the  parent  cell. 

ANILIN  DYES. — React  readily.  It  is  stained  by  slightly  modified 
Gram's  method.  Spores  double  stain  readily. 

GROWTH. — Is  very  rapid,  especially  on  glucose  media.  Requires 
anaerobic  conditions. 

Plates. — 0 >n  gelatin,  colonies  develop  in  2-3  days,  and  under  the  microscope  resemble 
those  of  the  Hay  bacillus.  As  they  become  larger  gas  bubbles  form.  On  agar  plates  at  37" 
the  colonies  appear  as  an  irregular,  dense  net-work  of  threads. 

Stab  culture.— In  gelatin,  growth  occurs  in  the  lower  part  of  the  tube;  the  gelatin  is 
liquefied,  gas  given  off  and  the  growth  settles  to  the  bottom.  Agar  cultures  are  torn  into 
several  parts  by  the  gas  which  is  formed.  In  the  liquid  on  the  bottom  of  the  tube,  giant 
whips  can  be  found  by  staining. 

Streak  culture. — On  agar,  offers  no  special  characteristics.  Grows  on  potato  with- 
out forming  a  scum. 

Bouillon. — Becomes  cloudy,  and  in  1-2  days  the  growth  settles  to  the  bottom  as  a 
low,  adherent  sediment,  and  in  a  few  days  the  liquid  becomes  clear. 

Glucose  gelatin,  colored  with  litmus.— In  air  at  37°  is  liquefied  and  litmus  first 
reduced,  then  in  presence  of  oxygen  it  becomes  red — acid  production. 

Milk.— Develops  a  good  growth;  a  part  of  the  casein  is  precipitated.  Starch  is  not 
changed  to  sugar. 

OXYGKN  REQUIREMENTS. — Is  an  obligative  anaerobe.  Grows  in  vacuum, 
hydrogen,  carbonic  acid,  etc. 

TEMPERATURE. — Growth  is  best  at  the  temperature  of  the  body. 
Can  grow  at  ordinary  temperature. 

BEHAVIOR  TO  GELATIN.  -Liquefies. 

AEROGENESIS.— On  glucose  media,  especially  when  distinctly  alka- 
line, it  gives  rise  to  the  production  of  g"as. 

ATTENUATION. — Bouillon  cultures  retain  virulence  for  months.  In 
general  the  virulence  varies  greatly.  Virulence  increased  in  mixed 
cultures  (prodigiosus,  proteus,  etc.). 

IMMUNITY. -One  attack  of  malignant  edema  does  not  protect 
ag-ainst  a  second.  100  c.c.  of  heated  or  filtered  cultures  injected  into 
guinea-pigs  in  three  portions  confers  immunity;  6-8  c.c.  of  the  serous 
exudate  accomplish  the  same  result. 

PATHOGEN ESIS. — Rabbit  susceptible — distinction  from  symptomatic 
anthrax.  The  horse,  hog",  dog,  cat,  chicken,  dove,  guinea-pig",  mouse 
and  man,  are  susceptible.  Cattle  are  immune.  Subcutaneous  inocu- 
lation in  guinea-pig's  of  %  c.c.  or  more  of  bouillon  culture  produces 
death  in  about  24  hours.  Marked  subcutaneous,  spreading,  reddish 
edema;  muscles  dark.  Bacilli  present,  single  or  in  threads,  in  subcu- 
taneous tissue,  on  serous  surfaces  as  peritoneum,  etc.;  scarce  or  not 
present  in  the  blood.  25-30  c.c.  of  the  filtered  bouillon  culture,  in- 
jected subcutaneously,  kills  guinea-pig's. 

INFECTION. — Takes  place  by  inoculation  through  wounds.  Poisoned 
arrows  of  the  New  Hebrides.  Rag-picker's  disease,  supposed  to  be  due 
to  inhalation  of  this  bacillus;  is  probably  anthrax. 

DIAGNOSIS.— To  be  distinguished  from  anthrax.  The  distribution, 
form,  motility  and  cultural  properties,  will  enable  differentiation. 

300 


DRAWINGS.  301 


Bacillus  CEdematis   Maligni,    No.  II,  Novy  (1893). 

ORIGIN. — From  guinea-pigs  inoculated  with  milk  nuclein  obtained 
from  casein  by  digestion  with  artificial  gastric  juice. 

FORM. — In  the  animal  body  it  occurs  usually  in  single  rods,  4-5 
times  as  long  as  wide;  may  also  occur  in  short  threads.  On  artificial 
media  it  develops  as  straight  or  bent  rods,  sometimes  forming  peculiar- 
ly twisted  threads.  The  contents  are  often  granular,  and  show  a 
"bright  body  at  one  end. 

MOTILITY. — Possesses  a  slight  swaying  motion,  which  is  often 
absent.  Has  lateral  flagella,  and  in  pure  culture,  as  well  as  in  the 
animal,  it  gives  rise  to  giant  whips  which  may  attain  a  length  of 
40-50-72  microns  (Fig.  7,  p.  39). 

SPORULATION. — Spore  formation  not  observed. 

ANILIN  DYES. — Stain  readily.     Gram's  method  applicable. 

GROWTH. — Depends  upon  the  vitality  of  the  organism.  When 
taken  from  an  animal  it  grows  rapidly. 

Plates. — On  glucose  ag-ar  good  colonies  develop  in  2-3  days  at  37°.  Show  a  very 
irregular,  fibrillated  border,  and  often  give  rise  to  gas  bubbles.  May  contain  giant  whips. 

Stab  culture.— Develops  only  in  the  lower  part  of  the  tube.  In  glucose  agar  having 
proper  alkalinity,  it  develops  rapidly  forming  a  plainly  visible  growth  along  the  line  of 
inoculation;  the  agar  is  soon  torn  into  several  parts  by  the  gas  that  is  produced.  Cultures 
soon  die  out.  ....  , 

Streak  culture. — Growth  on  glucose  agar  only  when  p \ygen  is  completely  excluded. 
It  forms  a  white  film  which  spreads  over  the  surface.  On  acid  agar  involution  forms  develop. 

Bouillon. — An  excellent  growth  develops  which  in  24  hours  settles  to  the  bottom  as  a 
loose,  flocculent  sediment;  the  liquid  above  becomes  clear— distinction  from  M.  edema  and 
S.  anthrax  bacilli. 

Glucose  gelatin,  colored  with  litmus.— Is  liquefied  and  acid  is  produced — the  litmus 
is  reduced  and  then  turned  red. 

OXYGEN  REQUIREMENTS.— Is  an  obUgatwe  anaerobe.  Grows  in  vacuum, 
hydrogen,  nitrogen,  carbonic  acid,  illuminating  gas. 

TEMPERATURE.— Does  not  grow  below  25°.  Optimum  temperature 
about  39°.  Can  withstand  freezing  for  24  hours. 

BEHAVIOR  TO  GELATIN. — Liquefies. 

AEROGENESIS.  — In  alkaline  media  gives  rise  to  gases.  Volatile 
acids,  as  butyric  acids,  etc.,  are  formed  in  artificial  culture  and  also 
in  the  body  of  rabbits. 

ATTENUATION.  —Cultures  left  in  hydrogen,  or  exposed  to  light,  lose 
their  virulence.  It  is  not  attenuated  when  kept  in  the  dark  or  when 
frequently  passed  through  animals.  Lost  virulence  can  be  reconsti- 
tuted by  inoculation  with  a  "mixed"  culture  containing  Proteus 
vulgaris. 

IMMUNITY. — Not  conferred  by  a  non-fatal  inoculation,  or  by  old, 
weakened  cultures,  or  by  the  serous  exudate  of  the  pleural  cavity. 

PATHOGENESIS. — Subcutaneous  injection  of  i  c.c.  of  hydrogen 
bouillon  cultures  kills  guinea-pigs,  rabbits,  white  rats,  white  mice, 
doves,  in  12-24  hours.  Marked  subcutaneous  edema  present;  serous 
exudates  in  thoracic  and  abdominal  cavities.  Cover-glass  prepara- 
tions made  from  the  subcutaneous  tissue  or  serous  surfaces,  as  peri- 
toneum, shows  usually  enormous  numbers  of  bacilli,  and  frequently 
giant  whips  are  also  present.  The  latter  are  visible  as  colorless  spirals. 

DIAGNOSIS.  —  The  morphological  characteristics  distinguish  it 
readily  from  malignant  edema  and  from  symptomatic  anthrax. 

302 


DRAWINGS.  303 


Bacillus  Tetani,  Nicolaier  (1884). 
TETANUS.  LOCK-JAW;  WUNDSTARRKRAMPF  (Germ.);  TETANOS  (Fr.). 

ORIGIN. — Found  in  animals  that  die  of  tetanus  after  inoculation 
with  earth;  in  traumatic  tetanus  of  man  and  animals;  in  head- 
tetanus;  tetanus  of  new-born.  Present  in  intestines. 

FORM. — Large,  narrow  rods  with  rounded  ends;  may  form  threads. 

MOTILITY. — Is  motile.     May  show  long-  spirals  or  giant  whips. 

SPORULATION.— Occurs  rapidly,  in  24-48  hours  at  37°.  Forms  ter- 
minal spores,  with  enlargement — drum-sticks. 

ANILIN    DYES. — Stain     rapidly.      Gram's    method    is    applicable. 

GROWTH. — This  is  slow. 

Plates. — At  ordinary  temperature  colonies  develop  in  gelatin  in  4-7  days,  and  resem- 
ble those  of  the  Hay  bacillus  or  of  Proteus.  The  gelatin  is  slowly  liquefied  and  gas  pro- 
duced. On  agar  plates  the  colonies  appear  as  faint  clouds  which,  under  the  microscope, 
are  seen  to  be  oval  and  partly  surrounded  by  a  whorl  of  threads  which  are  finer  than  those 
of  other  anaerobes. 

Stab  culture. — Development  restricted  to  the  lower  part  of  the  tube.  Cultures  in 
glucose  gelatin  tubes  show  along  the  line  of  inoculation  a  cloudy  growth,  radiating  outward 
into  the  surrounding  gelatin;  resembles  that  of  the  Root  bacillus.  Eventually  the  gelatin 
is  liquefied.  Gas  bubbles  present.  In  glucose  agar  at  37°  the  growth  is  sometimes  indis- 
tinct and  shows  radiations. 

Streak  culture. — On  glucose  agar  the  growth  is  rapid  and  is  practically  invisible. 

Bouillon.— At  37°  becomes  diffusely  cloudy  and  remains  so  for  several  days;  event- 
ually the  growth  settles  to  the  bottom,  forming  a  scarcely  visible  sediment — distinction  from 
the  preceding  anaerobes. 

Glucose  gelatin,  colored  with  litmus. — At  37°  becomes  permanently  liquefied;  a  very 
small  sediment  forms,  and  the  culture  remains  blue,  showing  absence  of  acid  formation — 
distinction  from  preceding. 

Milk. — It  grows  well  in  milk  without  inducing  any  change.  Does  not  invert  starch. 
The  growth  on  potato  is  invisible. 

OXYGEN  REQUIREMENTS. — It  is  an  obligative  anaerobe  and  grows  in 
vacuum,  hydrogen,  nitrogen,  and  carbonic  acid. 

TEMPERATURE.—  No  growth  below  16°.     The  optimum  is  about .38°. 

BEHAVIOR  TO  GELATIN. — Liquefies. 

AEROGENESIS.. — Gaseous  products;  disagreeable  odor;   ILS. 

ATTENUATION.— Partial   loss  of    virulence    by  culture. 

IMMUNITY. — Iodine  trichloride;  thymus  bouillon  cultures;  injec- 
tion of  filtered  culture;  of  purified  toxin;  blood-serum  of  artificially 
immunized  rabbits,  horse,  sheep,  dog;  milk  of  immunized  goat.  The 
nucleohiston  from  the  thymus  gland  destroys  the  tetanus  toxin. 

PATHOGENESIS. — Man,  horse,  sheep,  guinea-pigs,  young  cattle, 
goats,  white  mice  and  white  rats,  are  susceptible.  Rabbits  and  dogs 
are  less  susceptible.  Ducks  and  chickens  are  immune.  It  is  not  in 
the  blood  but  is  present  at  the  point  of  inoculation,  although  in  small 
numbers;  at  times  it  may  be  wholly  absent.  Intensely  poisonous  pro- 
ducts. The  filtered  bouillon  culture  in  a  dose  of  0.0002  c.c.  may  kill  a 
mouse,  and  0.002  c.c.  may  kill  a  guinea-pig.  The  strictly  pure  tetanus 
spores  cannot  produce  the  disease.  Mixed  infection. 

INFECTION. — Occurs  through  wounds.  Poisoned  arrows  of  the 
New  Hebrides  contain  tetanus  and  malignant  edema  spores. 

DIAGNOSIS. — The  detection  of  the  bacillus  is  difficult  because  of  its 
scarcity,  and  because  of  the  presence  of  other  anaerobic  and  aerobic 
bacteria.  The  pus  should  be  removed  from  the  wound  with  a  sterile 
drawn-out  glass  tube  pipette  and  transferred  to  glucose  litmus  gela- 
tin. A  loopful  of  this  dilution  should  be  transferred  to  each  of  8  or  10 
tubes  of  liquefied  glucose  agar.  These  are  then  poured  into  Petri 
dishes  and  developed  in  hydrogen.  The  characteristic  colony  is  oval, 
one  end  of  which  is  surrounded  by  a  whorl  of  threads. 

The  original  glucose  litmus  gelatin  is  developed  at  35°  and  a  por- 
tion is  then  injected  under  the  skin  of  a  guinea-pig,  or  a  white  mouse. 

A  portion  of  the  pus  should  be  stained  direct  and  examined  for 
the  long  slender  tetanus  bacilli  and  for  "drum-sticks." 

304 


DRAWINGS.  305 


306  BACTERIOLOGY. 

The  Culture  of  Anaerobic  Bacteria. 

Obligative  anaerobic  bacteria,  those  which  grow  only 
in  the  absence  of  oxygen,  require  special  conditions  for  cul- 
tivation. Their  growth  is  favored  by  the  addition  of  1  to  2 
per  cent,  of  glucose  to  the  nutrient  medium,  such  as  gela- 
tin,  bouillon  or  agar.  Freshly  prepared  media  are,  as  a 
rule,  best  adapted  for  cultural  purposes. 

The  numerous  methods  which  have  been  proposed  for 
obtaining  growths  of  anaerobic  bacteria  can  be  classified 
under  the  following  heads: 

1.— Exclusion  of  oxygen.  4. — Displacement  of  air. 

2. — Exhaustion  of  air.  5. — Cultures  apparently  in  air. 

3.— Absorption  of  oxygen.  6. — Microbic  association. 

Exclusion  of  oxygen.  — Several  different  methods  can  be 
grouped  under  this  head.  Thus,  the  earliest  method  em- 
ployed was  to  cover  the  liquid  with  a  layer  of  oil.  Although 
not  perfect,  yet  this  method  of  Pasteur  can  at  times  be  em- 
ployed to  advantage.  Later  on,  Koch  endeavored  to  obtain 
colonies  under  anaerobic  conditions  by  placing  a  thin  mica 
sheet  on  the  surface  of  a  gelatin  plate.  Better  results  are 
obtained  by  covering  the  gelatin  or  agar  plate  with  another 
sterile  glass  plate  (Sanfelice). 

The  well-known  method  of  Liborius,  culture  in  deep  lay- 
ers of  gelatin  or  agar,  depends  upon  the  exclusion  of  air. 
The  method  is  simple  and  very  convenient.  The  tubes 
should  contain  glucose  agar  or  gelatin,  and  the  medium 
should  be  about  2  inches  deep.  Stab  cultures  are  made  in 
the  usual  manner.  Growth  develops  along  the  line  of  in- 
oculation in  the  lower  two-thirds  of  the  medium,  while  the 
upper  third  (about  ^  inch),  serves  to  exclude  the  air.  In 
order  to  insure  complete  exclusion  of  oxygen,  the  contents 
of  an  ordinary  agar  or  gelatin  tube  can  be  liquefied  and 


THE  CULTURE   OF   ANAEROBIC    BACTERIA.  307 

then,  with  proper  precautions  against  contamination,  poured 
on  top  of  the  inoculated  medium  and  quickly  cooled.  This 
extra  layer  is,  as  a  rule,  unnecessary. 

Isolated  colonies  can  be  obtained  by  the  method  just  given.  The 
liquefied  glucose  agar  or  gelatin  tubes  are  inoculated  in  the  usual 
manner  for  making-  plates.  The  contents  of  the  tubes  are  then  solidi- 
fied and  an  extra  layer  of  the  medium  is  poured  into  each  tube. 

The  method  of  Roux  deserves  mention  under  this  head. 
The  inoculated  medium  is  drawn  up  into  a  sterile  glass-tube 
pipette  (Fig.  61),  which  is  then  sealed  above  and  below  the 
liquid  by  means  of  a  flame.  A  deep  layer  is  thus  obtained 
with  little  or  no  air  above.  By  cutting-  the  tube  a  colony 
can  be  easily  reached. 

Exhaustion  of  air. — Pasteur  in  his  studies  on  the  bacillus 
of  malignant  edema  employed  special  tubes  which  were 
connected  with  an  air-pump,  and,  when  a  vacuum  was 
reached,  they  were  sealed  in  the  flame.  The  method  has 
been  simplified  by  Gruber  and  is  easy  of  execution.  Special 
test-tubes,  with  a  constriction  below  the  cotton  plug1  may 
be  obtained.  After  the  medium  is  inoculated  a  vacuum  is 
produced  in  the  tube  which  is  then  sealed  in  the  flame.  If 
desired,  colonies  can  be  obtained  by  "  rolling-  "  the  sealed 
tube. 

Absorption  of  oxygen. — If  a  solution  of  pyrogallic  acid  is 
rendered  alkaline  it  will  immediately  become  dark,  then 
brown,  and  finally  black,  due  to  the  rapid  absorption  of 
oxygen.  This  reaction  has  been  utilized  in  a  variety  of 
methods.  Other  chemicals  may  be  used  for  absorbing  oxy- 
gen, but  they  have  no  advantage  over  that  mentioned. 
Buchner's  method  consists  in  placing  the  inoculated  tube 
inside  of  a  larger  one,  which  contains  on  the  bottom  some 
pyrogallic  acid.  Caustic  alkali  is  added  to  the  acid  and  the 
tube  is  then  closed  at  once  with  a  rubber  stopper. 


308 


BACTERIOLOGY. 


Displacement  of  air. — This  can  be  accomplished  by  pass- 
ing through  the  tube  or  apparatus  a  current  of  inert  gas. 
Hydrogen  is  employed  most  often  for  this  purpose.  Car- 
bonic acid  has  been  employed  by  Pasteur  and  others,  but 
it  is  not  as  indifferent  a  gas  as  hydrogen.  If  employed,  it 
should  be  washed  by  passage  through  a  solution  of  sodium 
carbonate.  Illuminating  gas  may  be  used  with  fair  results. 


FIG.  50.    Simple  bottle  for  anaerobes  (F.  G.  N.). 

The  Liborius  tube  which  is  most  often  mentioned  in  this 
connection  is  a  test-tube  with  a  constriction  below  the  neck. 
At  a  distance  of  about  2  inches  from  the  bottom  a  narrow 
side-tube  is  attached,  and  is  continued  on  the  inside  almost 
to  the  bottom  of  the  test-tube.  After  the  medium  is  inocu- 
lated a  current  of  hydrogen  is  passed  into  the  tube,  through 
the  side-tube,  until  the  air  has  been  displaced.  When  this 
result  is  attained  the  tube  is  sealed  below  the  cotton  plug 
and  finally  the  side-tube  is  likewise  sealed.  Various  modi- 
fications have  been  proposed,  but  although  they  render  the 


THE   CULTURE  OF  ANAEROBIC    BACTERIA.  309 

method  less  expensive,  they  do  not  materially  simplify  the 
procedure. 

Fig.  50  shows  an  apparatus  which  was  employed  at 
one  time  by  the  author.  It  can  be  constructed  by  any  one 
and  is  simple,  inexpensive,  and  will  give  excellent  results. 
Ordinary  test-tubes  are  employed  instead  of  the  expensive 
tubes  of  Liborius.  Moreover,  a  large  number  of  tubes  can 
be  placed  in  one  apparatus.  The  directions  for  use  as 
given  in  connection  with  the  author's  special  apparatus 
(p.  312),  are  applicable  to  this  bottle.  The  stop-cocks 
serve  to  seal  the  apparatus. 

A  great  variety  of  apparatus  has  been  described  for 
the  purpose  of  obtaining  colonies.  Some  of  these,  like 
Botkin's,  enable  one  to  make  use  of  the  ordinary  Petri 
dishes.  More  often,  however,  they  consist  of  a  special 
dish,  such  as  that  of  Kitasato. 

Cultures  in  the  presence  of  air. — This  method,  from  the 
nature  of  the  organisms  under  consideration,  would  seem 
to  be  impossible.  Nevertheless,  as  the  author  has  shown, 
good  cultures  can  be  obtained  without  the  use  of  any  spe- 
cial apparatus,  and  these  cultures  are  made  with  appar- 
ently free  access  of  air.  When  a  tube  of  glucose  agar 
is  liquefied,  and  then  allowed  to  become  solid,  a  drop  or 
two  of  water  will  separate  out  on  the  surface.  If  a  stab 
culture  is  now  made,  for  instance  of  symptomatic  an- 
thrax, it  will  show  a  good  growth  not  only  along  the  line 
of  inoculation  in  the  deeper  layers  of  the  agar,  but  also 
in  the  liquid  on  the  surface.  This  liquid  is  apparently 
in  direct  contact  with  the  air.  It  is  possible,  however, 
that  the  carbonic  acid  and  other  gases  given  off  by  the 
organism  displace  the  air  from  the  tube,  and  allow  thus 
a  growth  to  take  place.  It  may  be  remarked  in  this  con- 
nection that  this  growth  in  the  water  of  condensation  is 
extremely  rich  in  those  peculiar  spirals  known  as  giant- 
whips  (p.  39). 


310  BACTERIOLOGY. 

A  better  and  more  useful  procedure  than  the  above  is 
to  employ  gelatin  containing-  two  per  cent,  of  glucose  and 
colored  with  litmus.  The  ordinary  test-tubes  containing 
this  material  are  inoculated  and  placed  direct  in  the  incuba- 
tor at  37°. 

Although  the  gelatin  melts  and  apparently  there  is 
free  access  of  air,  yet  abundant  growths  of  all  the  anae- 
robic bacteria  can  thus  be  obtained.  Similar,  though  not 
as  constant  results  have  been  obtained  with  glucose  bouil- 
lon containing  two  per  cent,  of  gelatin.  The  viscosity  of 
the  liquid  undoubtedly  prevents  the  penetration  of  the  air. 
The  cultures  grown  in  litmus  colored  glucose  gelatin  pre- 
serve their  vitality  better  than  on  any  other  medium. 
Moreover,  they  possess  another  advantage  in  this  that  the 
growth  is  readily  accessible  for  transplantation  or  for 
study.  For  these  reasons  it  is  the  author's  usual  method 
for  keeping  stock  cultures  of  anaerobes. 

MicroMc  association. — The  soil  seems  to  be  the  natural 
habitat  of  the  anaerobic  bacteria.  And  yet,  of  all  places 
this  would  seem  to  be  the  least  adapted  for  their  growth 
owing  to  the  abundance  of  oxygen.  When  a  strongly 
aerobic  organism,  such  as  the  B.  prodigiosus  or  Proteus 
vulgaris,  is  inoculated  into  a  tube  of  bouillon,  and,  if  at  the 
same  time  an  anaerobic  organism  is  planted,  it  will  be 
found  that  both  bacteria  will  develop.  Apparently  the 
aerobic  form  consumes  the  oxygen  in  the  immediate  neigh- 
borhood of  the  anaerobic  organism,  and  thus  allows  the 
latter  to  develop.  Such  cultures  are  intensely  virulent. 
Conditions  of  this  kind  not  only  favor  the  growth  of  anae- 
robic bacteria  in  the  soil,  but  are  known  to  bring  on  dis- 
ease. Tetanus  or  lock-jaw  is  induced  in  this  way,  as  a 
result  of  such  microbic  associations. 

Of  the  several  methods  touched  upon  in  the  preceding 
summary  two  deserve  especial  attention  owing  to  their 
practical  usefulness.  In  these  the  ordinary  test-tubes  are 


THE  CULTURE  OF  ANAEROBIC    BACTERIA.  311 

employed  and  no  apparatus  is  necessary.  The  methods  re- 
ferred to  are:  stab  culture  in  deep  layers  of  gelatin  or  agar, 
and  cultivation  in  glucose  litmus  gelatin. 

The  various  forms  of  apparatus  indicated  above  and 
described  fully  in  most  of  the  text-books,  are  far  from 
being-  satisfactory.  The  use  of  special  tubes  or  of  special 
plates,  where  a  large  number  are  to  be  used,  is  a  matter  of 
considerable  expense.  The  treatment  of  each  tube  by 
itself  and  the  subsequent  sealing-  involves  a  waste  of  time, 
and  is  not  altogether  free  from  danger.  At  least  one  death 
is  recorded  as  the  result  of  endeavoring  to  seal  a  hydrogen 
culture  of  the  tetanus  bacillus.  It  is  obviously  desirable 
to  make  use  of  the  ordinary  test-tubes  and  ordinary  Petri 
dishes.  This  can  be  done  by  means  of  the  special  ap- 
paratus1 shown  in  Fig.  51-53.  During  the  past  six  years 
this  apparatus  has  been  in  constant  use  in  this  laboratory, 
and  the  fact  that  frequently  several  hundred  cultures  are 
made  by  the  students  at  one  time  will  indicate  its  practical 
usefulness. 

The  bottle  shown  in  Fig.  51  is  intended  for  tube  culture. 
It  is  made  in  two  sizes,  for  large  and  for  small  tubes.  The 
small  bottle  has  an  internal  diameter  of  8  cm. ,  and  the  in- 
side height  to  the  neck  should  be  16  cm.  The  large  bottle 
has  a  corresponding  diameter  of  10  cm.  and  a  depth  of  20 
cm.  It  is  provided  with  a  hollow  stopper  which  should  be 
not  less  than  4  cm.  in  diameter.  There  are  openings  in  the 
glass  stopper  corresponding  to  the  two  tubes  in  the  neck  of 
the  bottle.  One  of  these  openings  has  a  glass  tube  attached 
which  extends  down  to  within  a  short  distance  of  the  bot- 
tom. After  the  gas  has  been  passed  for  some  time,  the 
stopper  is  turned  through  an  angle  of  90°,  thus  effectually 
sealing  the  bottle. 

Ordinary  test-tubes  containing  glucose  bouillon,  gela- 
tin, agar  or  potato  are  inoculated  in  the  usual  manner.  The 
projecting  part  of  the  cotton  plug  is  cut  off  close  to  the 

1  Centralblatt  fur  Bakteriologie  U,  p.  581,  1893;  16,  p.  566,  1894. 


312  BACTERIOLOGY. 

mouth  of  the  tube,  and,  the  plug1  is  slightly  raised  with 
sterilized  forceps  in  order  to  facilitate  diffusion  of  the  gas. 
The  inoculated  tubes  are  then  placed  in  the  bottle  by  means 
of  a  pair  of  forceps  (Fig.  46),  and  the  apparatus  is  now  con- 
nected with  a  Kipp's  hydrogen  generator.  The  current  of 
hydrogen  shonld  be  passed,  first,  through  an  alkaline  solu- 
tion of  lead  acetate,  then  through  a  six  per  cent,  solu- 
tion of  potassium  permanganate,  and  finally  through  a  solu- 
tion of  silver  nitrate.  After  passing  through  the  apparatus 
the  gas  passes  through  a  small  wash-bottle  containing 
water,  which  serves  as  a  valve.  A  rapid  current  of  gas  is 
passed  through  the  bottle  for  1  to  2  hours.  The  bottle  is 
then  sealed  by  turning  the  stopper,  and  is  set  aside  in  the 
incubator. 

The  hydrogen  is  generated  from  ordinary  granulated  zinc  by 
means  of  commercial  sulphuric  acid.  The  stopper  should  not  be 
greased  with  vaselin,  but  with  a  mixture  of  bees-wax  and  olive  oil 
(1-4).  Before  applying  the  lubricant,  it  is  advisable  to  spread  a  layer 
of  cotton  over  the  bottom  of  the  flask.  Rubber  bands  should  be  slip- 
ped over  the  stopper  and  around  the  lateral  tubes.  This  is  to  prevent 
the  stopper  from  being  raised  by  the  pressure  of  the  confined  gases 
when  the  apparatus  is  placed  in  the  incubator. 

When  the  cultures  have  developed  they  should  be  taken  out  of 
the  bottle  and  preserved  the  same  as  ordinary  bacteria.  Care  should 
be  exercised  when  removing  the  stopper  so  as  to  avoid  breakage.  It 
should  first  be  turned  so  as  to  allow  air  to  enter.  At  the  same  time  the 
rubber  bands  are  removed.  The  thumb  and  forefinger  of  the  left  hand 
should  rest  firmly  on  the  shoulder  of  the  stopper,  while  this  is  grad- 
ually worked  to  and  fro  with  the  right  hand.  This  little  precaution 
will  prevent  the  sudden  jerking  out  of  the  stopper. 

The  pyrogallate  method  can  be  used  in  connection  with  this  bot- 
tle. 2-3  g.  of  the  acid  can  be  placed  on  the  bottom,  the  inoculated 
tubes  then  inserted,  and  finally  the  necessary  concentrated  alkali  can 
be  delivered  from  a  pipette.  The  stopper  should  then  be  inserted  at 
once  and  turned  at  right  angles. 

Fig's.  52  and  53  show  two  forms  of  apparatus  for  obtain- 
ing- plate  cultures.  The  former  is  provided  with  a  stopper 
like  that  in  the  bottle  just  described.  This  apparatus  can 


THE  CULTURE  OF  ANAEROBIC    BACTERIA.  313 

be  used  for  the  gas  or  for  the  pyrogallate  method.  The 
apparatus  shown  in  Fig  53  is  provided  with  a  slightly  dif- 
ferent stopper,  and  can  be  used  for  the  gas,  pyrogallate  or^ 
vacuum  method.  In  the  latter  case  the  wider  end  of  the 
stopper  should  be  provided  with  a  piece  of  rubber  tubing, 
which  should  be  clamped  tight.  The  apparatus  provided 
with  the  ordinary  stopper  (Figs.  51-52),  should  never  be 
used  for  vacuum  work  since  the  atmospheric  pressure  will 
cause  the  stopper  to  wedge  so  firmly  that  it  will  be  almost 
impossible  to  turn. 

The  lower  half  of  the  apparatus  should  have  an  internal  diameter 
of  12  cm.  and  an  inside  height  of  12  cm.  The  apparatus  can  hold  6  or 
S  Petri  dishes.  It  can  be  used  for  small  flasks,  or  for  a  large  number 
of  tube  cultures.  The  total  height  of  the  apparatus  should  not  ex- 
ceed 24  cm.  Each  flange  should  be  2  cm.  wide  and  f  cm.  thick.  The 
outer  circumference  should  be  ground  vertically  so  that  a  rubber  band 
can  be  employed  to  effectually  seal  the  apparatus.  It  is  necessary, 
therefore,  that  the  diameter  of  the  top  and  bottom  should  be  exactly 
the  same.  The  unground  surfaces  of  the  flanges  should  be  parallel  or 
nearly  so  in  order  that  the  clamps  will  not  slip  off. 

The  gelatin  or  agar  Petri  dishes  are  placed  in  the  lower 
jar.  It  is  unnecessary  and  undesirable  to  remove  the  tops. 
The  upper  part  is  then  placed  in  position  and  the  wide  rub- 
ber band  is  slipped  over  the  circumference.  Three  clamps 
are  then  applied  to  the  flanges.  A  slit  piece  of  rubber  tub- 
ing should  be  slipped  over  the  jaws  of  the  clamps.1  Hydro- 
gen is  then  passed  through  the  apparatus  in  the  manner 
described  on  p.  312.  Finally  the  stopper  is  turned  and  the 
apparatus  set  aside  for  the  organisms  to  develop. 

The  pyrogallate  method  can  be  used  with  excellent  re- 
sults. 3-5  g.  of  pyrogallic  acid  are  placed  in  a  glass  dish, 
which  is  about  10  cm.  in  diameter  and  about  3  cm.  high. 
This  is  placed  on  the  bottom  of  the  lower  jar  and  covered 
with  a  strip  of  glass  about  5  cm.  wide.  The  Petri  dishes 
are  then  stacked  on  top.  The  upper  half  of  the  apparatus 

1  No.  1.  Amateur  vise  made  by  the  Phoenix  Hardware  Mnf  g.  Co., 
Phoenix,  N.  Y. 


314 


BACTERIOLOGY. 


is  placed  in  a  position  so  that  only  a  narrow  slit  remains 

open.  By  means  of  a  pi- 
pette or  other  arrangement 
25  c.c.  of  concentrated 
potash  solution  (1:4),  are 
added  as  rapidly  as  possi- 
ble. The  top  is  then  closed 
completely  and  the  rubber 
band  and  clamps  applied 
as  before. 

Laboratory  work.  —  Liquefy 
four  glucose  agar  tubes  by  heat- 
ing in  the  water-bath;  then  allow 
to  solidify  in  an  upright  position. 
When  cool  make  deep  stab  cul- 
tures of  the  4  pathogenic  anae- 
robic organisms  and  then  place 
the  tubes  at  37°. 

If  the  agar  in  the  tube  is 
less  than  one  inch  deep,  it  will 


FIG.  52. 


FIG.  53- 


FIGS.  51-53.  The  author's  apparatus  for  the  culture  of  anaerobic  bacteria.  FIG.  51.. 
Bottle  for  tube  cultures.  FIG.  52.  Apparatus  for  Petri  dishes  or  tubes.— gas  or  pyrogal- 
late  method.  FIG.  53.  Apparatus  for  plates  or  tubes,— gas,  pyrogallate  or  vacuum  method. 


THE   CULTURE  OF   ANAEROBIC    BACTERIA. 


315 


be  necessary  to  pour  on  top,  after  inoculation,  the  contents  of  another 
agar  tube,  taking-  care  to  sterilize  the  mouths  of  both  tubes.    The  drop 
or  two  of  liquid  which  sometimes  accumulates  on  the  surface  of  the 
agar,  and  invariably  on  the  bottom  of    the  tubes  can 
be  stained  for  ordinary  flagella  and  for  giant-whips. 

Likewise   inoculate   litmus  glucose  gelatin  tubes 
and  place  at  37°. 


The  four  organisms  are  also  planted  in  glucose 
bouillon  and  these  tubes  are  then  placed  in  bottles  in 
hydrogen  at  37°.  In  about  3  days  the  tubes  can  be 
taken  out  and  examined  for  spores.  It  is  best  to  re- 
move the  bacterial  deposit  from  each  tube  by  means 
of  a  sterile  drawn-out  pipette  (Fig.  61).  The  growth 
can  then  be  placed  in  a  small  Esmarch  dish,  or  salt 
cellar,  and  after  dilution  with  water  it  can  be  used  for 
making  cover-glass  preparations.  When  preparations 
are  made  direct  from  the  bouillon  they  are  likely  the 
give  a  bad  back-ground  owing  to  the  organic  matter 
present.  The  spores  can  be  readily  double-stained. 

Inclined  glucose  agar  should  also  be  inoculated 
with  the  four  organisms,  and  the  culture  should  be  de- 
veloped in  hydrogen  at  37°  for  24  hours.  The  growths 
should  then  be  examined  carefully  in  hanging-drops 
for  motion,  spores,  giant-whips,  etc.  The  latter  will 
be  found  especially  abundant  in  the  liquid  of  conden- 
sation on  the  bottom  of  the  tube.  With  this  material 
cover-glass  preparations  will  be  made,  as  presently 
described,  and  employed  for  demonstrating  the  pres- 
ence of  flagella  or  whips. 


FIG.  $4.  Tube 
cultivation,  on  po- 
tato, of  the  tuber- 
cle bacillus;  with 

Glucose  agar  Petri  dishes  should  also  be  prepared,  open  capillary. 
Some  of  these  should  be  placed  in  the  plating  appara- 
tus (Fig.  52)  and  cultivated  in  an  atmosphere  of  hydrogen.      The 
pyrogallate  method  (p.  313)  should  be  used  to  develop  another  set  of 
the  plates.    A  third  set  of  plates  should  be  grown  in  a  vacuum  (Fig. 
53)  which  is  brought  about  by  means  of  a  Chapman  aspirator. 

Make  the  following  cultures  at  the  same  time  as  the  preceding: 

1. — Streak  cultures  of  the  tubercle  bacillus  on  inclined  glycerin 

agar  and  on  glycerin  potato  (Roux  tube,  Fig.  54).     A  pure  culture,  or 


316  BACTERIOLOGY. 

a  tubercle  from  a  guinea-pig  inoculated  with  tuberculosis  should  be 
used.  The  material  should  be  thoroughly  spread  over  the  surface  of 
the  medium. 

2.— Streak  cultures  of  the  aviary  tubercle  bacillus  on  inclined 
glycerin  agar  and  on  glycerin  potato  as  above. 

3. — Streak  culture  of  the  Achorion  Schonleinii  (the  fungus  of 
favus)  on  ordinary  inclined  agar. 

4— Streak  culture  of  Actinomyces  (the  fungus  of  lumpy-jaw)  on 
ordinary  inclined  agar. 

5. — Streak  culture  of  the  fungus  of  Madura-foot  on  inclined  agar 
or  glycerin  potato. 

After  these  inoculations  have  been  made  the  cotton  plug  of  each 
tube  is  cut  off  close  to  the  mouth  of  the  tube.  The  end  of  the  tube  is 
then  rapidly  turned  in  a  flame  till  the  cotton  changes  color.  The  tube 
is  then  sealed  either  with  a  rubber  cap  (previously  soaked  in  mercuric 
chloride),  or  with  sealing-wax,  or  with  paraffin  of  a  high  melting 
point,  56°.  Ordinary  corks  can  be  used  to  advantage  provided  they 
are  first  immersed  in  mercuric  chloride  solution  and  steamed  for  at 
least  i  hour.  The  heated,  slightly  charred  cotton  plug  is  pushed  into 
the  tube  by  means  of  sterile  forceps.  The  softened,  sterile  cork  is 
then  inserted.  The  sealed  tubes  are  then  placed  in  the  incubator  at 
39°  for  several  weeks. 

Instead  of  sealing  the  tubercle  cultures  air-tight,  it  is  advisable 
to  employ  a  cork  through  which  passes  a  drawn-out  capillary  tube 
{Fig.  54). 

The  Staining  of  Flagella. 


The  locomotive  organs  of  bacteria,  as  described  on  p. 
35,  are  extremely  thin,  wavy  whips  or  flagella.  These  can- 
not be  seen  in  the  unstained  preparations,  although  under 
favorable  conditions  they  can  be  demonstrated  by  photo- 
graphy. Only  very  rarely  can  motion  be  observed  in  the 
liquid  immediately  surrounding  the  organism.  In  simple 
stained  preparations  the  flagella  are  likewise  invisible,  in- 
asmuch as  they  do  not  readily  take  up  the  dye.  The  pres- 
ence of  whips  or  flagella  can,  therefore,  be  satisfactorily 
demonstrated  only  by  the  aid  of  special  staining  methods. 
The  procedure  as  employed  below  is  essentially  that  of 
Loftier. 


THE   STAINING  OF    FLAGELLA.  31 T 

In  order  to  obtain  good  stains  of  flagella  special  care 
must  be  given  to  the  preparation  of  the  cover-glasses. 
These  should  be  cleaned  according  to  the  method  giverLpn^ 
p.  140,  and  when  spread  must  contain  as  little  organic  mat- 
ter as  possible  in  order  to  prevent  the  formation  of  a  dirty 
precipitate  on  the  cover-glass.  .Excellent  cover-glasses 
can  be  made  by  resorting  to  dilution.  For  this  purpose,  a 
small  loopful  of  the  turbid  fluid  found  on  top  of  the  agar 
stab  culture,  or  at  the  bottom  of  the  inclined  agar  culture 
of  the  edema  bacillus  No.  II,  or  of  the  symptomatic  an- 
thrax bacillus,  is  transferred  to  a  large  drop  of  distilled 
water  on  a  glass  slide. 

By  means  of  a  straight  platinum  wire,  three  transfers 
are  made  from  this  drop  to  another  drop  of  distilled  water 
on  the  same  slide.  This  second  drop  will  now  contain  only 
a  small  number  of  bacteria  and  very  little  foreign  matter. 
By  means  of  a  platinum  wire,  with  a  very  small  loop,  not 
much  larger  than  a  pin-head,  transfers  can  now  be  made  to 
six  or  eight  clean  wide  cover-glasses.  Each  small  loopful 
is  spread  at  once  over  as  much  of  the  surface  of  the  cover- 
glass  as  possible.  The  thin  film  of  liquid  evaporates  al- 
most immediately,  and  the  cover-glass  can  then  be  fixed  by 
passing  it  once  through  the  flame.  Over-heating  the  cover- 
glass  is  very  likely  to  destroy  the  slender  flagella.  This 
can  be  prevented  if  the  cover-glass  is  held  between  the 
thumb  and  index  finger  as  it  is  passed  through  the  flame. 

The  cover-glass,  with  the  specimen  side  up,  is  held  in 
a  pair  of  forceps  and  covered,  by  the  aid  of  a  pipette,  with 
the  hot  mordant  solution.  The  cover-glass  is  then  held 
over  the  flame  for  about  a  minute.  The  flame  should  be 
turned  down  till  it  is  less  than  1^-2  inches  high.  The 
liquid  should  be  warmed  so  as  to  give  off  vapors,  but 
should  not  be  actually  boiled.  As  fast  as  evaporation 
takes  place  fresh  mordant  solution  should  be  added,  and 
at  no  time  should  it  be  allowed  to  dry  down  on  the  cover- 
glass. 


318  BACTERIOLOGY. 

After  one  or  two  minutes  of  careful  heating  the  mor- 
dant is  then  thoroughly  and  completely  washed  off  the 
cover-glass  by  a  jet  of  water.  This  can  be  easily  done  if 
the  specimen  has  not  been  over-heated.  If  the  edge  has 
dried  down,  it  should  be  loosened  with  a  pin  or  knife  and 
then  washed  off.  To  still  farther  clean  the  cover-glass,  it 
may  be  dipped  for  a  few  seconds  in  absolute  alcohol  and 
again  washed  with  water. 

The  excess  of  water  is  drained  from  the  cover-glass 
which  is  then  covered  with  a  hot  saturated  solution  of 
anilin-water  fuchsin.  The  specimen  is  then  gently  and 
slowly  heated  over  the  flame  for  one  to  two  minutes,  avoid- 
ing actual  ebullition.  It  is  then  heated  to  boiling  for  about 
half  a  minute,  and  finally  it  is  thoroughly  washed  with 
water  and  examined  with  the  A  inch  oil  immersion  objective. 
A  number  of  slender  flagella  can  be  seen  surrounding  each 
cell. 

Summary  for  staining  flagella: 

Dilution  cover-glass  preparation. 

Dry  in  air. 

Once  through  flame. 

Mordant,  hot  (1  to  2  min.). 

Wash  in  water  (and  clean). 

Dip  in  alcohol  (few  seconds). 

Rinse  in  water. 

Anilin-water  fuchsin,  hot  (1  to  2  min.). 

Wash  in  water  and  examine. 

Dry  in  air. 

Mount  in  balsam. 

The  mordant  employed  is  a  slight  modification  of  that  originally 
proposed  by  Loffler.  It  has  the  following  composition  (A.  Fischer): 

Dry  tannin 2  g. 

Water 20  c.c. 

Ferrous  sulphate  (1:2) 4  c.c. 

Cone.  ale.  fuchsin 1  c.c. 

The  tannin  should  be  kept  in  a  desiccator.  It  is  dissolved  in 
the  stated  amount  of  water  by  the  aid  of  gentle  heat.  The  two  other 


THE  STAINING  OF    FLAGELLA.  319 

solutions  are  then  added  and  the  liquid  is  filtered  at  once.  A  volu- 
minous precipitate  must  remain  on  the  filter,  otherwise  the  mordant 
is  useless.  The  mordant  can  be  used  at  once,  and  will  keep  for  six 
weeks  or  more.  It  should  be  kept  in  the  dark.  The  mordant  is  said 
to  give  excellent  results  with  all  motile  bacteria. 

The  stain  employed  is  made  by  adding-  4  to  5  g.  of  fuch- 
sin  to  100  c.c.  of  anilin  water,  to  which  1  c.c.  of  a  1  per 
cent.  NaOH  solution  has  been  added.  Hot,  freshly  pre- 
pared carbolic  fuchsin,  or  an  aqueous  1  per  cent,  solution 
of  fuchsin,  may  be  used.  Both  mordant  and  stain  should 
be  kept  warm  while  in  use.  The  iron  plate  shown  in  Fig. 
22  (p.  150),  will  be  found  useful  for  this  purpose. 

Giant-whips. — These  interesting-  forms  are  especially 
abundant  in  the  few  drops  of  condensation  water 
which  accumulate  at  the  bottom  of  the  freshly  inclined 
agar.  They  are  met  with  under  these  conditions  in 
cultures  of  most  of  the  known  motile  bacteria.  Owing  to 
their  extremely  large  size  they  can  readily  be  seen  in  the 
hanging-drop.  The  author's  method  is  to  place  on  a  cover- 
glass  a  drop  of  blood-serum,  which  is  then  inoculated  with 
the  turbid  liquid  mentioned.  In  about  15  minutes,  more  or 
less  agglutination  of  the  bacteria  results  and  the  large 
spiral  bodies  are  then  plainly  visible.  They  may  be  broad, 
spindle-shaped,  or  very  long,  slender,  wavy  lines.  They 
have  been  observed  to  exceed  100  /j.  (shs  inch)  in  length  (see 
p.  38). 


Bacillus  Leprae,    Hansen  (1879). 

LEPROSY    BACILLUS. 

ORIGIN. — It  is  found  in  enormous  numbers  in  the  leprous 
nodules  of  the  skin;  to  less  extent  in  the  mucous  membrane, 
lymphatic  glands,  liver,  spleen,  marrow,  etc.  It  usually 
occurs  in  masses  within  the  so-called  leper  cells.  It  may 
be  present  in  the  blood  in  small  numbers. 

FORM. — Small,  narrow  rods,  which  resemble  the  tubercle 
bacillus. 

MOTILITY. — It  is  non-motile. 

SPORULATION. — Bright  bodies,  are  frequently  observed 
within  the  cell,  but  as  in  the  case  of  the  tubercle  bacillus, 
these  are  not  spores. 

ANILIN  DYES. — In  fresh  material  the  bacilli  can  be  readily 
stained  with  the  ordinary  anilin  dyes.  They  can  likewise 
be  easily  demonstrated  in  such  material  by  means  of  the 
method  employed  for  staining-  the  tubercle  bacillus  (p.  324). 
The  latter  stain  fails  when  the  tissue  has  been  kept  in  alco- 
hol for  some  time.  In  that  case  Gram's  method  will  give 
excellent  results  (Chapter  XV). 

GROWTH. — This  has  not  been  obtained  on  artificial  media 
with  certainty.  It  is  to  be  considered  as  a  typical  obliga- 
tive  parasitic  organism. 

PATHOGENESIS. — The  constant  presence  of  this  bacillus  in 
enormous  numbers,  in  leprous  tissue  justifies  the  prevailing 
view  that  it  is  the  cause  of  that  disease.  Nevertheless,  it 
should  be  remembered  that  as  yet  unquestioned  pure  cul- 
tures have  not  been  obtained,  and  hence,  successful  inocu- 
lations are  impossible.  Direct  infection  with  leprosy  tissue 
has  given  but  few,  if  any,  positive  results.  The  monkey, 
cat,  dog,  goat,  hog,  chicken,  guinea-pig,  rabbit,  etc.,  are 
apparently  not  affected. 

INFECTION. — Leprosy,  as  in  the  case  of  tuberculosis,  may 
be  congenital.  As  a  rule,  however,  it  is  acquired  but  the 
exact  mode  of  infection  is  as  yet  unknown.  It  would  seem 
that  leprosy,  like  glanders,  attacks  first  of  all  the  nasal 
mucous  membrane.  The  leprosy  bacillus  leaves  the  body 
with  the  secretions  of  the  nose  and  mouth. 

DIAGNOSIS. — The  bacillus  of  leprosy  is  to  be  distinguished 
from  that  of  tuberculosis.  The  inoculation  of  a  guinea-pig 
will  decide  the  presence  of  tubercle  bacilli.  An  examina- 
tion of  the  nasal  secretion,  and  of  sputum  should  be  made 
in  suspected  cases. 

320 


DRAWINGS.  321 


21 


Bacillus  Tuberculosis,  Koch  (1882). 

TUBERCLE   BACILLUS. 

ORIGIN.— In  tuberculosis  of  mammals;  in  lupus  vulgaris.  The 
bacillus  of  chicken  tuberculosis  is  distinct  from  that  of  mammals. 

FORM. — Very  narrow,  rather  long-  rods  which  are  smaller  than  the 
diameter  of  a  red  blood  cell.  They  may  be  beaded.  The  ends  are  dis- 
tinctly rounded  and  the  bacillus  itself  may  be  straight,  or  more  fre- 
quently is  slightly  bent  or  nicked.  Occurs  usually  single,  but  may 
form  short  threads  of  3-6  cells.  In  the  sputum,  tissues  etc.,  it  is  fre- 
quently found  in  small  bunches.  Rarely,  it  occurs  in  branching-  form 
and  with  club-shaped  ends. 

MOTILITY. — Has  no  motion. 

SPORULA.TION. — Frequently  shows  a  number  of  bright  bodies  within 
the  cell,  but  these  cannot  be  considered  as  true  spores  (p.  32).  The 
bacillus  itself  possesses  a  relatively  high  power  of  resistance  to  heat, 
desiccation,  acids,  putrefaction  etc. 

ANILIN  DYES. — It  stains  very  slowly  and  difficultly  with  simple  ani- 
lin  dyes;  readily  with  hot  carbolic  fuchsin,  or  anilin-water  fuchsin  or 
gentian  violet.  When  once  stained  it  is  difficult  to  decolor,  whereas 
ordinary  bacteria  dp  so  readily — distinction  from  ordinary  bacteria. 
Gram's  method  applicable,  but  requires  along  exposure  to  the  dye. 

GROWTH. — Takes  place  very  slowly,  so  that  usually  several  weeks 
elapse  before  it  becomes  clearly  visible.  Furthermore,  a  special  tem- 
perature, at  or  near  that  of  the  body,  and  special  media  as  blood- 
serum  or  glycerin-agar,  etc.,  are  necessary.  Access  of  air  is  also  es- 
sential. The  cultures  have  a  characteristic  yeast-like  odor.  It  is 
possible  to  accustom  the  tubercle  bacillus  to  grow  on  ordinary  bouil- 
lon and  agar,  even  at  or  near  the  room  temperature-  attenuation. 

Plates. — No  growth  has  been  obtained  on  plates.  Colonies  can  be  readily  obtained 
by  making  successive  streaks  on  glycerin-agar  or  blood-serum.  Colonies  obtained  direct 
from  the  sputum  are  round,  white,  opaque,  and  raised,  resembling  colonies  of  white  yeast. 
On  subsequent  culture  the  colonies  form  dry,  grayish  scales.  Under  the  microscope  they 
appear  as  interwoven,  twisted  strands  of  threads. 

Stab  culture. — Can  be  obtained  on  glycerin-agar.  The  growth  is  restricted  to  the  up- 
per part  of  the  tube.  It  spreads  over  the  surface  as  a  thick,  raised  plaque  which  at  first  is 
white,  but  later  becomes  yellowish. 

Streak  culture. — On  'glycerin-agar  or  blood-serum  it  develops  an  abundant  dry,  gran- 
ular, raised  growth,  which  at  first  is  grayish,  but  later  takes  on  a  light  yellow  tinge. 

Potato. — In  Roux  tubes  containing  $  per  cent,  of  aqueous  glycerin  (Fig.  54,  p.  315) 
the  growth  is  thick,  yellowish  and  in  ridges.  Requires  at  least  2  weeks  at  39°. 

Bouillon. — One  which  contains  5-6  per  cent,  of  glycerin  is  necessary.  A  dry  folded 
growth  covers  the  surface;  the  liquid  remains  clear  and  a  granular  sand-like  deposit  forms. 
The  material  planted  must  float  on  the  surface  of  the  liquid,  otherwise  the  culture  will  not 
develop,  or  but  very  poorly.  Such  bouillon  cultures,  filtered  and  concentrated,  constitute 
the  so-called  tuberculin. 

OXYGEN  REQUIREMENTS. — Free  access  of  oxygen  is  necessary  for 
growth.  It  is  a  facultative  anaerobe  (Frankel). 

TEMPERATURE. — Optimum  at  37-39°.  Slight  variations  above  or 
below  this  stop  the  growth.  It  cannot  grow  at  room  temperature. 

BEHAVIOR  TO  GELATIN. — No  growth  at  ordinary  temperature.  It 
does  not  peptonize  blood-serum. 

ATTENUATION. — Prolonged  artificial  cultivation  results  in  partial 
attenuation.  This  is  especially  marked  when  grown  on  potato  and 
similar  media.  Passage  of  the  bacillus  through  doves  or  frogs  yields 
an  attenuated  form  which  may  grow  at  ordinary  room  temperature. 
The  tubercle  bacillus  as  found  in  man  possesses  a  variable  virulence. 

PATHOGENESIS. — See  p.  325.     DIAGNOSIS.— See  p.  324. 

INFECTION. — Takes  place  most  frequently  along  the  respiratory 
tract — Inhalation  tuberculosis.  May  occur  through  wounds—  Inoculation 
tuberculosis,  and  also  through  food — Intestinal  tuberculosis.  In  the  latter 
case  the  bacilli  introduced  into  the  intestines  may  localize  in  distant 
parts  of  the  body.  Placental  infection  may  occur. 

322 


DRAWINGS.  323 


324  BACTERIOLOGY. 

Recognition  of  the  Tubercle  Bacillus. 

Ziehl-Neelsen  method. — A  large  loopful  of  the  sputum  is 
transferred  to  a  wide  cover-glass  and  thoroughly  spread 
over  the  surface.  It  is  then  allowed  to  dry  in  the  air  or 
by  moving  it  to  and  fro,  over  the  flame,  after  which  it  is 
fixed  in  the  usual  way.  The  cover-glass  is  held  in  the 
forceps,  specimen  side  up,  and  covered  with  carbolic  fuchsin 
solution.  It  is  warmed  over  a  low  flame  for  1  to  2  minutes, 
avoiding  actual  ebullition.  The  dye  should  not  be  allowed 
to  dry  down  on  the  cover-glass.  A  drop  or  two  of  the  stain 
should  be  added,  from  time  to  time,  to  prevent  this  from 
happening.  The  excess  of  dye  is  then  washed  off  with 
water  and  the  specimen  is  placed  in  dilute  nitric  acid  for 
10  to  15  seconds.  This  dilute  acid  solution  is  prepared  by 
adding  3  or  4  drops  of  the  concentrated  acid  to  a  watch- 
glass  full  of  water.  When  the  color  disappears  it  is  at  once 
transferred  to  dilute  alcohol  (60  to  70  per  cent.),  where  it  is 
moved  about  till  it  is  almost  decolored.  After  this  it  is 
washed  with  water  and  stained  for  a  few  seconds  with 
methylene  blue.  The  latter  is  washed  off  with  water  and 
the  specimen  is  then  examined  with  the  Ath  inch  oil  immer- 
sion objective.  It  should  show  the  bright  red  tubercle 
bacillus  on  a  light  blue  back-ground.  The  ordinary  bac- 
teria that  may  be  present  are  stained  likewise  blue.  It  is 
evident,  therefore,  that  the  ordinary  bacteria  are  readily 
decolored  by  treatment  with  acid  and  alcohol  and  in  this 
respect  differ  from  the  tubercle  bacillus.  This  is  due  to  a 
difference  in  the  chemical  composition  or  to  a  concentra- 
tion of  the  protoplasm  of  the  tubercle  cell,  rather  than  to 
the  presence  of  a  denser  cell-wall. 

Heavily  stained  preparations  can  be  obtained  by  float- 
ing the  prepared  cover-glass  on  slightly  warmed  carbolic 
fuchsin  solution  for  15  to  30  minutes,  then  decoloring  as 
above. 


THE  TUBERCLE  BACILLUS.  325 

Summary  of  the  method  given: 

Cover-glass  preparation.  Dilute  alcohol  (60  per  cent.). 

Dry  in  air.  Wash  in  water. 

Fix  in  flame.  Methylene  blue  (i  - i  min.). 

Carbolic  fuchsin,  hot,  (1-2  min.).  Wash  in  water  and  examine. 

Wash  in  water.  Dry  in  air. 

Dilute  nitric  acid  (10-15  seconds).  Mount  in  balsam. 

The  sputum  should  be  collected  in  the  morning  immediately 
after  rising,  inasmuch  as  at  this  time  it  is  likely  to  be  rich  in  bac- 
teria. It  may  be  necessary  at  times  to  instruct  the  patient  that 
sputum,  and  not  saliva,  is  what  is  wanted.  The  material  should  be 
poured  into  a  wide  glass  dish,  such  as  that  of  Petri,  and  examined  for 
the  presence  of  yellowish  particles.  These  are  portions  of  the 
caseous  matter  from  the  lungs  and  are  likely  to  be  rich  in  bacteria. 
The  sputum  should  be  examined  also  for  elastic  fibers,  and  the  pres- 
ence of  streptococci  and  of  other  bacteria  should  be  noted  (mixed 
infection). 

Suspected  urine  should  be  centrifugated  and  the  deposit 
examined  in  the  same  way  as  indicated  above.  In  the  case 
of  milk,  it  is  advisable  to  centrifugate  and  then  to  inject 
the  combined  cream  and  sediment  into  guinea-pigs. 

Occasionally  sputum,  pus,  or  other  products  of  disease 
will  not  reveal  the  tubercle  bacillus  by  this  method. 
Either  the  germ  is  present  in  a  spore-like  condition, 
or  it  is  present  in  very  small  numbers  and  thus  escapes 
detection.  In  such  a  case  it  is  necessary,  as  with  urine  or 
milk,  to  resort  to  an  animal  experiment  in  order  to  estab- 
lish the  diagnosis.  For  this  purpose  some  of  the  material 
is  injected  into  the  peritoneal  cavity  of  a  guinea-pig.  In 
three  or  four  weeks  the  animal  is  killed,  and  if,  on  post- 
mortem examination,  tubercular  nodules  are  found  in  the 
abdominal  cavity  they  should  be  examined  for  the  tubercle 
bacillus.  A  portion  of  the  cheesy  matter  fromithe  inside  of 
the  nodule  should  be  used  for  the  examination.  Cultures 
may  be  made  as  indicated  on  p.  315. 

PATHOGENESIS. — Man,  monkey,  cattle,  horse,  ass,  hog,  goat,  par- 
rot, guinea-pig,  field  mouse,  rabbit,  and  cat  are  susceptible.  White 


326  BACTERIOLOGY. 

mice,  rats,  canaries,  and  dogs  are  somewhat  insusceptible.  The  pig- 
eon, sparrow,  chicken,  and  cold  blooded  animals  are  immune.  Inocu- 
lation of  pure  cultures  produces  tuberculosis  in  susceptible  animals. 
The  bacilli  are  usually  very  abundant,  but  at  other  times  are  scarce 
and  difficult  to  find.  Guinea-pig's  are  extremely  susceptible  to  the  in- 
traperitoneal  injection  of  the  tubercle  bacillus,  whereas  rabbits  are 
somewhat  less  susceptible.  There  is  a  marked  loss  in  weight  and  death 
may  result  in  10-14  days.  If  the  dose  is  small  this  will  not  occur  for 
from  4  to  6  weeks,  or  later.  The  peritoneum  may  be  studded  with  fine 
sand-like  tubercles.  Larger  tubercles  are  present  in  the  liver,  spleen 
and  lungs.  Very  large  tubercles  are  usually  present  on  the  omentum . 
These  have  a  soft,  caseous  center  and  are  rich  in  bacilli. 

The  diagnosis  of  tuberculosis  in  cattle  is  effected  by 
the  injection  of  a  small  dose  of  tuberculin.  A  marked  rise 
in  the  temperature  occurs,  and  may  persist  for  a  day  or 
more. 

The  staining  reaction  as  given  above  will  usually  be 
sufficient  to  establish  the  presence  of  the  tubercle  bacillus. 
In  doubtful  cases,  the  animal  experiment  must  be  resorted 
to.  It  should  be  borne  in  mind,  however,  that  a  few  bac- 
teria are  known  which  resist  decoloration  by  an  acid  and 
hence  may  be  mistaken  for  the  tubercle  bacillus.  More- 
over, the  mere  presence  of  nodules  or  tubercles  in  an  animal 
does  not  imply  the  presence  of  the  tubercle  bacillus  since 
pseudo-tuberculoses  are  known  which  are  due  to  several  differ- 
ent kinds  of  bacteria.  These  possible  contingencies  may 
be  briefly  alluded  to. 

1. — The  leprosy  bacillus  stains  like  the  tubercle  bacillus. 
It  can  be  stained  with  the  ordinary  dyes.  Moreover,  it 
exists  in  masses  and  cannot  be  transferred  to  animals,  nor 
can  it  be  grown  artificially.  The  recognition  of  tubercu- 
losis in  a  leper  can  be  effected  by  means  of  the  animal 
experiment.  Bacilli  resisting  decoloration  with  acids  have 
been  grown  from  leprosy  material. 

2. — The  smegma  bacillus  which  may  be  found  in  the 
smegma  of  the  prepuce  or  of  the  vulva,  stains  the 
same  as  the  tubercle  bacillus.  While  it  is  resistant  to 


THE  TUBERCLE   BACILLUS.  327 

acids,  it  is   readily  decolored  by  absolute   alcohol.     The 
smegrna  bacilli  of  diverse  origin  show  unequal  resistance 
to  decoloration  with  acids.     It  is  not  transferable  to  ani- 
mals and  is  very  difficult  to  cultivate.     The  animal  experi 
ment  will  ag^ain  demonstrate  the  tubercle  bacillus.     The 
smegrna  bacillus  should  be  expected,   whenever  urine  or 
secretions  of  the  g^enito-urinary  apparatus  are  examined. 
The  same  or  a  similar  organism  has  apparently  been  found 
in  the  mouth,  and  in  lung1  gangrene. 

To  differentiate  from  the  tubercle  bacillus  the  specimen  which 
has  been  stained  with  carbolic  fuchsin  should  be  decolored  by  immer- 
sion for  some  minutes  in  a  saturated  absolute  alcoholic  solution  of 
methylene  blue;  or  by  an  acid  alcoholic  solution  (alcohol  97,  HC1  3)  for 
10  minutes.  Another  procedure  is  to  treat  it  with  absolute  alcohol 
for  at  least  three  hours,  then  with  five  per  cent,  chromic  acid  for  at 
least  15  minutes,  after  which  it  is  stained  with  carbolic  fuchsin,  de- 
colored by  exposure  to  dilute  sulphuric  acid  for  2-3  minutes,  and 
finally  stained  with  concentrated  methylene  blue. 

In  order  to  cultivate  the  smegma  bacillus  it  is  advisable  to  ino- 
culate a  tube  of  liquefied  agar,  cooled  to  50°,  with  the  suspected  ma- 
terial. By  means  of  a  sterile  syringe  about  2  c.c.  of  blood  should  be 
drawn  from  the  vein  in  the  arm  (see  Chapter  XIV),  and  this  is  then 
mixed  with  the  agar.  The  blood  agar  mixture  is  poured  into  a 
sterile  Petri  dish,  which  is  then  set  aside  for  a  day  or  two  at  37°. 
The  colonies  should  be  examined  for  bacilli  which  resist  decolor- 
ation by  acids. 

3. — From  milk,  butter,  timothy  hay,  cow-dung",  etc., 
bacteria  have  been  isolated  which  stain  like  the  tuber- 
cle bacillus,  and  have  indeed  been  mistaken  for  this 
organism.  Guinea-pigs,  as  a  rule,  survive  injection 
whereas  rabbits  appear  to  be  immune.  The  inoculated 
guinea-pig's  may  die  in  a  month  or  two.  Numerous  small 
nodules  or  tubercles  are  present  in  the  mesentery,  peri- 
toneum, liver,  lung's,  etc.  Cultures  made  from  these  tuber- 
cles develop  in  three  or  four  days  and  apparently  atten- 
uate rapidly.  The  tubercles  do  not  show  giant  cells,  or 
caseation.  It  is  evident,  therefore,  that  in  some  instances 
it  is  necessary  to  supplement  the  cover-glass  examination 
by  a  histological  study  of  the  suspected  tubercles.  Bacteria 


328  BACTERIOLOGY. 

resisting  decoloration  with  an  acid  have  been  found  in  cer- 
tain eye  affections. 

4. — Pseudo-tuberculoses. — The  preceding  case  may  be 
said  to  fall  under  this  head.  There  are,  however,  bacteria 
which  give  rise  to  tubercles  or  nodules  but  otherwise  are 
easily  distinguished  from  the  tubercle  bacillus.  They  are 
easily  cultivated  and  stain  with  the  ordinary  dyes,  but  do 
not  give  the  double  stain  like  the  tubercle  bacillus. 
Pseudo-tuberculoses  are  rare  in  man,  though  rather  com- 
mon in  animals  (sheep,  chicken,  rabbits,  etc.). 

5. — Aviary  tuberculosis. — The  bacillus  present  in  the  or- 
dinary bird  tuberculosis  stains  like  the  tubercle  bacillus. 
It  grows  more  rapidly  on  various  media  and  forms  a  moist 
soft  growth,  which  unlike  that  of  the  tubercle  bacillus,  will 
easily  form  a  cloudy  suspension  when  stirred  into  water. 
Guinea-pigs  when  injected  subcutaneously  react  with 
a  local  affection,  whereas  intraperitoneal  injection  into 
a  chicken  yields  general  tuberculosis.  The  rabbit  is 
susceptible  to  both  organisms.  The  tubercles  do  not  show 
giant- cells.  The  bacillus  can  grow  at  a  higher  tempera- 
ture than  the  tubercle  bacillus,  but  otherwise  the  growths 
on  the  various  media  are  very  much  alike.  The  aviary 
tubercle  bacillus  may  naturally  infect  man,  rabbits,  and 
other  animals.  It  has  always  been  believed  to  be  a  variety 
of  the  mammalian  tubercle  bacillus.  Cultures  in  collodium 
sacs  seem  to  prove  that  the  aviary  bacillus  can  be  trans- 
formed into  that  of  mammals. 

6. — Fish  tuberculosis. — This  organism  was  isolated  from 
a  small  tumor  on  a  carp.  The  presence  of  giant  cells  and 
the  histological  structure  pointed  to  tuberculosis.  The 
bacillus  present  stained  like  that  of  tuberculosis,  but  was 
culturally  different.  It  grows  best  at  23-25°  and  does  not 
grow  at  39°.  It  would  seem  as  if  the  mammalian  tubercle 
bacillus  on  passage  through  cold-blooded  animals  (fish  and 
frogs)  gave  rise  to  a  variety,  as  in  the  case  of  bird  tuber- 
culosis. 


GLANDERS.  329 

Glanders. 

Diagnosis. — The  glanders  bacillus  is  extremely  difficult  ~ 
to  detect  in  old,  and  even  in  new  diseased  tissue.  Conse- 
quently, direct  detection,  as  in  the  case  of  tuberculosis, 
cannot  be  made  with  any  degree  of  certainty.  The  failure 
in  staining,  and  even  in  direct  cultivation,  renders  it  neces- 
sary to  resort  to  the  animal  experiment  in  doubtful  cases. 
For  this  purpose  two  procedures  are  available. 

1. — Method  of  Straus. — The  suspected  matter  or  secretion  is  dilu- 
ted with  sterile  water,  or  bouillon,  and  injected  intraperitoneally 
into  male  guinea-pigs.  The  reaction  is  manifested  in  a  few  days  by 
a  marked  swelling  of  the  testicles.  The  scrotum  becomes  red  or 
violet  and  adherent,  so  that  the  testicles  cannot  be  pushed  back  into 
the  abdomen.  Death  occurs  in  a  week  or  two.  Bacilli  may  be  isolated 
by  making  agar  streaks  from  the  spleen.  They  are  especially  abund- 
ant in  the  altered  testicles.  The  marked  swelling  of  the  organs, 
which  is  observed  within  three  or  four  days,  is  taken  as  confirming 
the  diagnosis  of  glanders. 

2. — Injection  of  mallein. — The  heated  and  filtered  bouillon  culture 
•of  the  glanders  bacillus,  when  injected  into  a  healthy  animal,  pro- 
duces no  rise  of  temperature,  and  at  most  a  slight  swelling  which  dis- 
appears within  24  hours.  In  a  glandered  animal  the  local  swelling  is 
extensive  and  painful;  the  lymphatic  vessels  which  lead  from  this 
part  are  likewise  enlarged  and  painful.  The  swelling  increases  for 
24-36  hours,  lasts  for  several  days,  and  finally  gradually  subsides  in 
about  8  or  10  days.  The  general  condition  of  the  animal  is  likewise 
markedly  changed.  A  marked  stupor  and  profound  prostration  is 
observed.  The  temperature  rises  1.5-2.5°  and  reaches  its  maximum 
between  the  10th  and  12th  hour.  A  diagnosis  can  thus  be  established 
within  48  hours. 

The  two  tests  should  always  be  carried  out  together,  inasmuch 
as  each  one  by  itself  is  subject  to  fallacy.  Thus,  the  Straus  reac- 
tion is  obtained  in  ulcerative  lymphangitis  of  the  horse.  This  dis- 
ease resembles  farcy  but  is  due  to  a  different  organism.  The  diseased 
animal  is  not  affected  by  mallein.  On  the  other  hand,  an  injection  of 
mallein  may  give  rise  to  fever  in  non-glandered  animals.  In  such 
cases,  however,  the  local  reaction  is  absent.  It  is  evident,  therefore, 
that  a  positive  Straus  reaction  and  negative  mallein  test;  or,  a  nega- 
tive Straus  reaction  and  increased  temperature  with  mallein,  would 
-exclude  glanders. 


Bacillus  Mallei,  Loffler  and  Schutz  (1882). 
GLANDERS:  MORVE  (jFV.);  ROTZ  (6rerm.);  MALLEUS  (Lat.). 

ORIGIN.— Found  in  the  nodules,  ulcers,  discharges,  etc.,  of  glan- 
ders or  farcy. 

FORM. — Rods  with  rounded  ends,  straight  or  slightly  curved, 
shorter  and  thicker  than  the  tubercle  bacillus.  Usually  single;  may 
grow  in  pairs  or  in  short  threads. 

MOTILITY. — It  shows  very  marked  Brownian  motion. 

SPORULATION.— Bright  bodies  are  frequently  found  in  the  cells,  a& 
in  the  tubercle  bacillus;  are  considered  by  Loffler  as  the  first  indica- 
tion of  degeneration.  Real  spores  are  unknown.  The  bacillus  itself 
is  not  very  resistant  to  desiccation. 

ANILIN  DYES. — It  is  stained  unevenly  and  decolors  rapidly.  Car- 
bolic fuchsin,  or  alkaline  anilin  gentian  violet,  or  anilin  fuchsin  stain 
well,  especially  when  warmed.  It  is  not  stained  by  Gram's  method. 

GROWTH. — This  occurs  best  at  a  relatively  high  temperature. 
Growth  is  rapid.  Glycerin  agar  is  the  best  medium. 

Plates. — As  a  rule,  colonies  cannot  be  obtained  with  gelatin.  On  glycerin  agar  at  37° 
excellent  colonies  form  in  a  day  or  two.  These  are  round,  grayish  and  glistening  in  ap- 
pearance, with  granular  contents  and  smooth  sharp  borders. 

Stab  culture. — Can  be  made  in  glycerin  agar;  in  gelatin  it  develops  very  slowly. 

Streak  culture. — On  glycerin  agar  forms  a  thick,  moist,  slimy,  semi-transparent 
growth.  On  potato  the  growth  is  very  characteristic.  At  first  it  forms  a  thin,  transparent, 
honey  or  amber-colored  growth  which  later  becomes  reddish-brown.  On  blood-serum  it 
forms  yellowish,  transparent  spots  which  eventually  fuse  together  and  yield  a  slimy,  whit- 
ish growth. 

Bouillon. — In  this  it  grows  readily  and  abundantly;  diffuse  cloudiness  with  slimy 
ring  on  the  surface.  Mallein  is  the  filtered  bouillon  culture  of  the  glanders  bacillus. 
It  is,  therefore,  analogous  to  tuberculin. 

In  milk  it  produces  an  acid  reaction. 

OXYGEN  REQUIRKMKNTS.  -It  is  a  facultative  anaerobe. 

TEMPERATURE. — It  does  not  grow  below  25°  very  readily,  or  above 
42°.  The  optimum  is  about  37°. 

BEHAVIOR  TO  GELATIN.- Scarcely  any  growth  at  first.  Eventually,, 
may  become  accustomed  to  growth  at  the  ordinary  room  temperature.. 

ATTENUATION.— This  takes  place  rapidly  when  grown  on  artificial 
media.  The  bacillus  must  be  frequently  passed  through  an  animal, 
otherwise  the  virulence  is  lost  and  the  organism  may  die  out. 

IMMUNITY.  — Small  amounts  of  a  bouillon  culture  injected  intra- 
venously into  dogs  confer  immunity. 

PATHOGENESIS.— Man,  horse,  ass,  guinea-pigs,  field  mice,  cats  and 
goats  are  highly  susceptible.  Ordinary  and  white  mice,  cattle  and 
hogs  are  immune,  while  dogs,  rabbits  and  sheep  are  but  slightly  sus- 
ceptible. White  mice  become  susceptible  when  fed  with  phloridzin. 
Susceptible  animals  on  inoculation  develop  typical  glanders.  In 

fuinea-pigs  death  results  in  4  to  6  or  8  weeks.     Field  mice  die  in  a  few 
ays.     Enlarged  lymphatics,  nodules  in  liver,  spleen,  etc.     Bacilli 
present. 

INFECTION. — This  may  occur  through  wounds— inoculation  glanders. 
In  one  instance  a  man  was  accidentally  and  fatally  inoculated  with  a 
pure  culture.  Probably,  the  usual  source  of  infection  in  horses  is- 
along  the  respiratory  tract. 

330 


DRAWINGS.  331 


Bacillus  Diphtherise,  Klebs,  Loffler  (1883). 

BACILLUS  OF  DIPHTHERIA. 

ORIGIN. — Found  in  diphtheric  pseudo-membranes,  and  in  very 
•small  numbers  in  the  spleen,  liver,  etc.,  of  diphtheria;  rarely  in  the 
throats  of  healthy  children. 

FORM.  Rather  large,  thick  rods  which  are  straight  or  slightly 
bent  or  wedge-shaped  and  have  rounded  ends.  The  form  is  subject  to 
considerable  variation,  and  rods  with  swollen,  club-shaped  ends  are 
frequently  met  with;  likewise  branching  forms— involutions.  Usually 
single;  length  variable. 

MOTILITY.— It  has  no  motion. 

SPORULATION.— Spores  have  not  been  observed.  The  bacillus  is 
very  susceptible  to  desiccation,  or  to  heat  of  50°  and  above. 

ANILIN  DYES. — Simple  anilin  dyes  react  poorly.  Round  polar 
bodies  and  transverse  bands.  Can  be  stained  best  with  carbolic 
fuchsin,  or  with  Loffler's  alkaline  methylene  blue.1  It  is  also  stained 
by  Gram's  method. 

GROWTH. —Very  rapid, especially  on  glycerin  agar  and  blood-serum. 

Plates. — On  gelatin  plates  kept  at  about  24°  it  forms  very  small  round,  white  colonies, 
which  have  granular  contents  and  irregular  borders;  do  not  liquefy  gelatin.  On  glycerin 
agar  plates,  kept  in  the  incubator,  excellent  colonies  form  in  24  hours.  The  deep  colonies 
are  round,  or  oval,  coarsely  granular.  The  surface  colonies  are  flat,  grayish  white,  glisten- 
ing, with  irregular  borders;  coarsely  granular  contents. 

Stab  culture.— In  gelatin  the  growth  spreads  over  the  surface  while  along  the  punc- 
ture only  a  very  limited,  scarcely  perceptible  growth  of  small,  round,  white  dots  occurs. 
Marked  involution  forms  present.  Vitality  is  retained  best  in  gelatin. 

Streak  culture.— On  glycerin  agar  a  thin,  grayish,  spreading,  adherent  film,  which 
is  quite  characteristic.  On  potato  the  growth  is  invisible  or  forms  a  dry,  thin  glaze — 
irregular  forms  of  the  bacillus  are  numerous.  On  blood-serum  it  forms  a  thick  white, 
opaque  growth;  branching  forms  may  be  present,  especially  if  the  medium  is  soft. 

Bouillon. — May,  or  may  not  be  diffusely  clouded;  the  growth  rapidly  subsides,  form- 
ing a  granular  deposit  on  the  sides  of  the  tube  and  on  the  bottom.  A  pellicle  usually  forms 
on  the  surface  and  the  liquid  becomes  perfectly  clear.  The  reaction  becomes  acid,  owing 
to  the  presence  of  muscle  sugar;  in  a  few  days  becomes  alkaline.  No  gas  or  indol  is 
produced.  Milk  is  not  altered. 

OXYGEN  REQUIREMENTS. — Facultative  anaerobe;  grows  best  in  air. 

TEMPERATURE.— Very  slight  growth  at  20°.  The  maximum  is 
about  42°  and  the  optimum  is  at  35-37°. 

BEHAVIOR  TO  GELATIN.— It  does  not  liquefy. 

ATTENUATION. — Cultures  isolated  direct  from  membranes  show 
marked  variation  in  virulence.  By  artificial  culture,  especially  if 
transplanted  infrequently,  the  virulence  is  still  further  diminished. 
Attenuated  by  growth  at  40°  in  a  current  of  air;  by  desiccation. 

IMMUNITY.— This  is  produced  by  filtered  bouillon  cultures  heated 
to  60-70°.  or  unheated,  Also  by  injections  of  thymus  bouillon  cultures 
previously  heated  to  65-70° .  Partial  results  with  IC13.  Living  cultures 
can  be  used.  Serum  of  artificially  immunized  animals  is  antitoxic. 

PATHOGENESIS. — Mice  and  rats  are  wholly  immune.  Finches,  spar- 
rows, doves,  chickens,  rabbits,  guinea-pigs  and  cats  are  susceptible. 
Likewise,  horses,  cattle,  dogs  and  goats.  Subcutaneous  inoculation 
in  guinea-pigs  usually  produces  death  in  24-48  hours.  Pseudo-mem- 
branous masses  form  at  point  of  inoculation;  an  extensive  hem- 
orrhagic  edema  forms  under  the  skin  and  exudates  occur  in  the  pleural 
cavity.  Inoculation  in  the  trachea  of  cats,  chickens,  doves,  rabbits 
etc.,  is  followed  by  pseudo-membrane  formation,  and  by  death.  In 
some  animals  as  rabbits,  typical  diphtheric  paralysis  of  the  extremities 
can  be  observed.  Local  swelling  and  scar,  in  prolonged  cases.  The 
filtered  bouillon  culture  contains  a  highly  poisonous  toxin  (p.  84). 

INFECTION. — This  undoubtedly  occurs  through  the  air,  or  by  con- 
tact with  infected  articles.  DIAGNOSIS.— See  p.  334. 

i  This  is  prepared  by  adding  30  c.c.  of  cone.  ale.  solution  of  methylene  blue  to  looc.c. 
•of  a  o.oi  per  cent,  solution  of  potassium  hydrate. 

332 


DRAWINGS. 


334  BACTERIOLOGY. 

Diphtheria. 

Diagnosis. — It  must  not  be  supposed  that  all  cases 
diagnosed  as  diphtheria  contain  the  Lsffler  bacillus.  It 
may  happen  that  diphtheria-like  conditions,  due  to  other 
organisms,  will  present  themselves.  This,  so-called  false 
diphtheria  is  frequently  due  to  streptococci;  at  times,  to 
staphylococci  or  to  various  bacilli.  A  correct  diagnosis  of 
true  diphtheria,  that  due  to  the  L5mer  bacillus,  can  there- 
fore be  made  only  by  the  aid  of  the  microscope.  Further- 
more, a  case  of  diphtheria  may  be  a  source  of  danger,  even 
after  complete  recovery,  because  of  the  continued  presence 
of  Lsffler  bacilli  in  the  mouth.  It  is  important,  therefore, 
not  only  to  recognize  the  disease  as  early  as  possible,  but 
also  to  recognize  the  time  when  the  infected  individual 
ceases  to  harbor  the  bacillus  in  question. 

In  order  to  carry  out  a  bacteriological  diagnosis  of 
diphtheria  it  is  necessary  to  have  some  cotton  swabs  and 
some  tubes  of  solidified,  inclined  blood-serum.  The  swab 
can  be  prepared  by  twisting  a  little  absorbent  cotton  around 
the  end  of  a  stout  wire  (2  mm.  wide  and  14  cm.  long).  The 
swabs  are  placed  in  plugged  test-tubes  and  sterilized  in  a 
dry-heat  sterilizer  (p.  160).  The  culture  medium,  usually 
employed,  is  known  as  L^ffler's  blood-serum.  It  is  pre- 
pared according  to  the  directions  given  in  Chapter  XIV. 
If  necessary,  ordinary  blood-serum  or  even  glycerin-agar 
may  be  used. 

The  sterile  swab  is  thoroughly  rubbed  over  the  affected  area, 
and,  if  possible,  a  portion  of  the  false  membrane  should  thus  be  re- 
moved. The  swab  is  then  streaked  over  the  surface  of  one  or  two  of 
the  sterile,  inclined  serum  tubes.  These  are  then  set  aside  in  the 
incubator  at  35-39°  for  18-24  hours.  The  use  of  a  disinfecting-  solu- 
tion, previous  to  the  swabbing  of  the  throat,  should  be  avoided. 

A  direct  microscopical  examination  can  be  made  by  rubbing-  the 
swab  over  one  or  more  cover-glasses.  These  are  then  fixed  and 
stained  with  Loffler's  methylene  blue  (p.  332).  As  a  rule,  however,  the 
most  satisfactory  results  will  be  obtained  by  examining  and  staining 
the  colonies  that  develop  on  the  blood-serum.  On  this  medium,  the 
Lomer  bacillus  outgrows  the  other  bacteria  that  may  be  present,  and 


DIPHTHERIA.  335 

hence  forms  large  colonies  which  have  a  very  characteristic  appear- 
ance. 

The  diphtheria  colonies,  as  they  appear  on  serum  within  24 
hours,  are  relatively  large,  round,  in  outline,  grayish  and  moist 
in  appearance.  The  center  is  thicker  and  more  opaque  than  the 
outer  zone  which  is  surrounded  by  a  slightly  wavy  border.  Cover- 
glass  preparations  made  from  these  colonies  and  stained  with 
Loffler's  methylene  blue,  will  reveal  the  characteristic  diphtheria 
bacillus.  Care  must  be  taken  not  to  over-stain  the  specimen  which, 
moreover,  should  be  examined  with  the  oil  immersion  objective. 
The  presence  of  irregular,  club-shaped  or  swollen  rods,  among  other- 
wise normal  bacteria,  is  an  important  mark  of  recognition.  More- 
over, many  of  these  rods  will  show  irregularities  in  staining.  Thus, 
transverse,  alternately  dark  and  light  bands  may  be  seen.  In  many 
cells,  one  or  more  bright  blue,  deeply  stained,  roundish  bodies  will  be 
observed.  These  characteristics  are,  as  a  rule,  sufficient  to  establish 
the  identity  of  the  organism  in  question.  It  is  well  to  ascertain  the 
kind  of  organisms  which  are  associated  with  the  Loffler  bacillus. 

In  doubtful  cases,  Gram's  stain  and  cultivation  on 
agar  at  low  temperature  may  be  resorted  to.  The  subcu- 
taneous injection  of  a  guinea-pig  with  ^  c.c.  of  a  24-48  hour 
bouillon  culture  will  serve  to  distinguish  virulent  from  non- 
virulent  forms,  and  from  the  pseudo-diphtheria  bacillus. 

The  pseudo-diphtheria  or  xerosis  bacilli  are  found  in  the 
mouth,  nose,  and  on  the  conjunctiva  of  healthy  persons, 
and  consequently  may  be  met  with  in  various  throat  and 
lung  affections.  They  grow  better  than  the  Lsffler  bacillus 
on  agar  and  impart  to  it  a  dark  color.  One  species  of  this 
group  forms  spores.  The  absence  of  pathogenic  properties 
is  an  important  distinction  from  the  diphtheria  bacillus. 
It  must  be  borne  in  mind,  however,  that  attenuated  diph- 
theria bacilli  may  be  met  with.  Indeed,  Roux  and  his  co- 
workers  even  at  the  present  time,  regard  this  organism  as 
a  modified,  weakened  diphtheria  bacillus.  An  important, 
though  not  absolute  distinction,  is  the  change  in  the  reac- 
tion of  litmus-colored  bouillon  cultures.  The  diphtheria 
bacillus  promptly  produces  an  acid  reaction,  whereas  the 
pseudo-diphtheria  bacillus  does  not.  Again,  the  diphtheria, 
like  the  tubercle  bacillus,  does  not  readily  form  a  homo- 
geneous suspension  when  touched  into  a  drop  of  water, 
whereas  the  pseudo -forms  do. 


336  BACTERIOLOGY. 

Apparently,  a  useful  method  of  distinguishing  the  Lomer  bacil- 
lus from  the  pseudo-diphtheria  bacillus  is  that  of  Neisser.  The  cul- 
tures are  developed  for  10-20  hours  on  the  Loffler's  serum  at  a  tem- 
perature not  exceeding  35° .  The  cover-glass  preparations  are  treated 
for  1-3  seconds  with  an  acetic  acid  methylene  blue  solution.  This 
is  prepared  as  follows:  1  g.  of  Griibler's  methylene  blue  is  dissolved  in 
20  c.c.  of  96  per  cent,  alcohol,  and  to  this  950  c.c.  of  distilled  water 
and  50  c.c.  of  glacial  acetic  acid  are  added.  The  specimen  is  rinsed 
in  water,  and  then  treated  for  3-5  seconds  with  aqueous  Bismarck 
brown.  The  latter  is  made  by  dissolving  2  g.  of  vesuvin  in  1  liter  of 
boiling  water.  By  this  process  the  isolated  polar  granules  are  ren- 
dered manifest  in  the  case  of  the  true  diphtheria  bacillus,  but  are 
not  to  be  seen  in  the  pseudo-diphtheria  bacilli.  According  to  Fraen- 
kel,  an  organism  which  fails  to  show  these  double  stained  polar  gran- 
ules is  not  a  true  diphtheria  bacillus.  On  the  other  hand,  pseudo- 
diphtheria  bacilli  may,  though  very  rarely,  show  slight  polar  bodies. 
In  such  cases  the  production  of  acidity  in  bouillon  and  the  effect  on 
guinea-pigs  should  be  tested. 


Pneumonia. 

The  Diplococcus  lanceolatus  is  of  extreme  interest  be- 
cause of  its  peculiar  biological  properties,  and  because  of  its 
frequent  occurrence  in  various  inflammatory  diseases.  Thus, 
it  is  by  far  the  most  common  cause  of  croupous  or  fibrinous 
pneumonia,  and  fully  one-half  of  the  cases  of  cerebro- 
spinal  meningitis  are  due  to  it.  Many  cases  of  broncho- 
pneumonia  are  likewise  ascribed  to  the  Fraenkel  diplo- 
coccus.  The  lungs  and  the  meninges  are,  therefore,  espe- 
cially suitable  for  its  development.  The  serous  surfaces, 
moreover,  furnish  a  favorable  location  and  hence  it  is  that 
this  diplococcus  is  a  frequent  cause  of  pleurisy,  pericar- 
ditis, endocarditis  and  peritonitis.  It  is,  likewise,  met  with 
in  inflammation  of  the  middle  ear  (otitis  media),  and  in 
abscesses. 

The  fact  that  it  is  frequently  present  in  the  healthy 
mouth,  nose  and  throat,  indicates  that  the  natural  resist- 
ance of  the  body  must  be  overcome  or  lowered  before  the 
organism  is  capable  of  inducing  a  disease.  Invasion  may 
occur  through  the  blood  or  through  the  lymph  spaces. 
The  virulence  of  the  germ,  the  way  in  which  it  enters,  and  the 


PNEUMONIA.  337 


place  of  least  resistance,  are  important  factors.  As  a  result, 
the  disease  may  be  acute  or  chronic,  wide-spread  or  local- 
ized. Infection  from  the  mouth  or  nose  may  lead  to  otitis 
or  to  meningitis,  especially  in  early  life;  whereas 


monia  is  more  common  in  old  age. 

ATTENUATION.  —  Cultures  from  different  sources  show  marked 
difference  in  virulence.  Cultures  isolated  from  pneumonia  in  the 
early  stage  are  more  virulent  than  those  obtained  later.  When 
grown  on  artificial  media  it  rapidly  attenuates  and  soon  dies  out, 
unless  it  is  passed  through  a  susceptible  animal,  as  a  rabbit,  every 
few  weeks.  Attenuation  in  a  few  days  at  42°.  The  virulence  and 
vitality  can  be  best  preserved  by  drawing  up  the  heart-blood  of  an 
infected  rabbit  into  sterile  tube  pipettes  (Figs.  61  d,  62  a).  These  are 
then  sealed  so  as  to  leave  as  little  oxygen  as  possible  in  the  tube. 
The  blood-serum  of  rabbits  is  well  adapted  for  growing  the  germ  and 
for  maintaining  its  virulence. 

Diagnosis.  —The  fact  of  the  frequent  presence  of  the 
diplococcus  in  the  normal  saliva  must  be  borne  in  mind 
when  examining  the  sputum  of  suspected  pneumonia. 
Moreover,  it  may  happen  that  the  diplococcus,  which  is 
very  abundant  in  the  early  stages  of  pneumonia,  may  be 
rare  or  even  absent  from  the  later  stages.  The  white 
mouse,  or  the  rabbit,  may  be  given  an  intra-peritoneal  in- 
jection of  the  suspected  material.  Or,  the  latter  may  be 
injected  subcutaneously  into  the  ear  of  a  rabbit.  In  this 
case  ordinary  sputum  will  not  kill,  whereas  that  of  pneu- 
monia will  prove  fatal  in  two  or  three  days,  or  later. 

A  microscopic  examination  of  the  sputum  or  exudates. 
in  man,  or  of  the  heart-blood  of  the  animal  should  show 
the  typical  capsulated,  lance-shaped  diplococcus,  which 
can  be  easily  stained  by  Gram's  method.  Glycerin  agar 
plates  should  be  developed  at  20°  and  at  37°.  The  Fraenkel 
diplococcus  will  not  grow,  or  only  very  exceptionally,  at  20°. 

Meningitis  may  be  induced  by  other  organisms  than  the  pneu- 
mococcus.  Among  these,  especially  deserving  attention,  is  the  Diplo- 
coccus  intracellularis  meningitidis,  which  in  many  respects  resembles  the 
gonococcus.  The  biscuit-shaped,  flattened  diplococci,  which  occur  in 
groups  or  within  cells,  do  not  stain  by  Gram's  method.  Unlike  the 

gonococcus,  they  can  be  readily  cultivated  on  agar  at  37°. 
22 


Micrococcus    Pneumoniae    Crouposae, 

Sternberg-,    Pasteur  (1880).     Fraenkel  (1886). 

FRAENKEL'S  DIPLOCOCCUS,  D.  LANCEOLATUS,  D.  PNEUMONIAS,  MICROBE 

OF    SPUTUM    SEPTICEMIA. 

ORIGIN. — Occasionally  in  the  saliva  and  nose  of  healthy  persons; 
especially  frequent  in  the  "rusty"  sputum  of  pneumonia.  The  same 
organism,  or  scarcely  distinguishable  varieties,  may  be  present  in 
cerebro-spinal  meningitis,  pleuritis,  peritonitis,  pericarditis  etc. 

FORM.— Occasionally  round,  usually  oval,  barley,  or  lance-shaped 
diplococci.  It  may  form  chains  of  4-6  cells,  and  hence  may  resemble 
a  streptococcus.  Owing  to  its  oval  form  it  is  sometimes  regarded  as 
a  bacillus.  In  the  animal  body,  it  is  surrounded  by  large  capsules 
(Fig.  5,  p.  29).  These  are  not  present  on  artificial  media. 

MOTILITY. — It  has  no  motion. 

SPORULATION  . — Unknown. 

ANILIN  DYES. — Stain  readily;  so  does  Gram's  method.  The  cap- 
sules remain  colorless. 

GROWTH. — Takes  place  somewhat  slowly  and  only  at  higher  tem- 
perature, and  on  alkaline  media.  The  ordinary  media  are  far  from 
being  favorable  for  its  growth.  This  is  seen  in  the  more  or  less 
marked  change  in  form  and  size,  and  especially  in  the  rapid  loss  of 
virulence  and  vitality.  5  per  cent,  glycerin  agar  makes  an  excellent 
medium.  2-3  per  cent,  of  glucose  is  also  beneficial.  It  should  be 
transplanted  every  two  or  three  weeks. 

Plates. ! — On  gelatin  plates  kept  at  24°,  small,  round,  sharply  defined,  slightly  granu- 
lar, whitish  colonies  develop  slowly.  On  agar  plates  in  the  incubator,  in  48  hours,  the  sur- 
face colonies  appear  as  delicate,  glistening,  transparent  drops  which,  under  the  microscope, 
are  round,  sharply  bordered,  finely  granular,  and  usually  possess  a  dark  center. 

Stab  culture.— In  gelatin  a  row  of  small,  white  granules  develop  along  the  line  of  ino- 
culation. Vitality  preserved  for  some  time;  acid  reaction.  Slight  growth  on  the  surface. 
Does  not  liquefy;  resembles  in  this  respect  the  streptococci. 

Streak  culture. — On  agar  in  the  incubator,  the  growth  develops  as  a  thin  layer  of 
delicate,  glistening,  almost  transparent  drops.  It  dies  out  rapidly.  On  blood-serum  it 
forms  a  transparent  film  of  dew-like  drops.  No  growth  on  potato. 

Bouillon. — Slight  diffuse  growth  which  soon  settles  yielding  a  very  slight- deposit. 

Milk. — Is  a  favorable  culture  medium;  it  may,  or  may  not  coagulate. 

OXYGEN  REQUIREMENTS.— It  is  a  facultative  anaero.be. 

TEMPERATURE.  -Growth  occurs  only  between  24  and  42°.  Its  opti- 
mum is  about  37°.  After  repeated  cultivation  it  may  adapt  itself  to 
a  temperature  of  about  18°. 

BEHAVIOR  TO  GELATIN. — Does  not  liquefy. 

ATTENUATION. — See  p.  337. 

IMMUNITY. — This  can  be  obtained  by  intravenous  injection  of  a 
very  small  amount  of  the  virulent  culture;  by  injections  of  filtered 
cultures,  especially  when  heated  to  60°;  blood  serum  of  immune  ani- 
mals has  a  questionable  value.  Blood-serum  from  pneumonic  pa- 
tients has  been  said  to  .imnfunize  rabbits  against  the  pure  culture, 
but  this  is  doubtful.  Injections  of  nuclein  immunize  rabbits. 

PATHOGENESJS. — In  rabbits  a  subcutaneous  injection  of  0.1-0.2  c.c. 
of  recently  isolated  bouillon  cultures  produces  death  in  24-48  hours. 
The  diplococcus  is  found  in  the  blood  and  internal  organs  and  is  sur- 
rounded by  a  capsule.  Tracheal  injections  in  rabbits  produce  true 
pneumonia.  Mice  and  rabbits  are  highly  susceptible;  guinea-pigs, 
sheep,  dogs  and  cats  are  less  susceptible.  Chicken  and  doves  are 
immune. 

i  Make  glycerin  agar  Petri  dishes  from  the  peritoneal  exudate  in  a  rabbit.  Make 
cover-glass  preparations  from  the  peritoneum,  surface  of  intestines  and  from  the  heart- 
blood,  and  stain  by  the  simple,  and  by  Gram's  method. 

338 


DRAWINGS.  339 


Bacillus  Pneumoniae,   Friedlander  (1883). 
FRIEDLANDER'S  PNEUMOCOCCUS,  OR  PNEUMO-BACILLUS. 

ORIGIN. — It  is  frequently  found  in  normal  saliva;  also  in  the  lungs 
and  in  the  "rusty"  sputum  of  pneumonia.  May  occur  elsewhere  as  in 
otitis.  It  has  been  found  in  the  air  and  in  water. 

FORM. — In  the  exudates  from  the  body  it  may  appear  as  an  oval 
coccus,  but  in  reality  it  is  a  short,  thick  rod,  which  may  grow  in  pairs 
and  even  in  short  threads.  In  the  animal  body  it  is  enveloped  by  a 
well-marked  capsule.  This  is  not  present  in  artificial  cultures.  - 

MOTILITY. — It  has  no  motion. 

SPORULATION. — Spores  have  not  been  observed.  Cultures  retain 
their  vitality  for  many  months. 

ANILIN  DYES.— The  cell  is  stained  readily  but  the  capsule  remains 
colorless.  The  bacillus  is  not  stained  by  Gram's  method — distinction 
from  Frankel's  diplococcus. 

'      GROWTH. — Is  rapid  and  abundant. 

Plates.!— On  gelatin  plates  it  develops  rapidly.  The  deep  colonies  are  round  or  oval, 
sharply  bordered;  finely  granular  and  yellowish.  The  surface  colonies  are  quite  character- 
istic and  appear  as  thick,  moist,  glistening,  white  masses  which  do  not  tend  to  spread  but 
rather  tend  to  become  convex  and  raised.  This  appearance  is  more  marked  when  grown 
at  15°  than  when  developed  at  25°.  In  the  latter  case,  they  may  be  flat  and  irregular.  No 
liquefaction. 

Stab  culture.— Growth  takes  place  along  the  entire  line  of  inoculation  and  is  espec- 
ially developed  on  the  surface  forming  a  "  nail-shaped"  culture.  As  the  culture  becomes 
old  the  gelatin  near  the  surface  becomes  brownish  in  color  and  small  gas  bubbles  may  form. 

Streak  culture.— On  ag-ar,  it  forms  a  thick,  mucus-like,  whitish  or  transparent,  moist, 
slimy  growth.  On  blood-serum  develops  as  a  grayish,  slimy  mass.  On  potato  it  forms  a 
thick,  yellowish,  sticky  growth,  which  shows  gas  bubbles.  A  diastatic  ferment  is  present. 

Boiiillon  is  rendered  diffusely  cloudy. 

Milk  is  not  coagulated,  or  but  rarely.    Indol  is  not  produced. 

.        OXYGEN  REQUIREMENTS.— It  is  a  facultative  anaerobe. 

TEMPERATURE.— It  grows  rapidly  even  at  low  temperatures,  16-20°: 
best  growth  in  the  incubator. 

BEHAVIOR  TO  GELATIN.— It  does  not  liquefy. 

AEROGENKSIS. — Abundant  production  of  gas  in  4  per  cent,  gelatin. 
Potato  cultures  grown  in  the  incubator  also  give  rise  to  gas. 

PATHOGENESIS. — It  is  pathogenic  for  mice  and  young  rats.  Guinea- 
pigs,  rabbits  and  dogs  are  less  susceptible.  The  virulence  varies 
considerably. 

The  Friedlander  bacillus  is  unquestionably  capable  of  inducing 
pneumonia.  However,  it  is  only  in  a  relatively  small  percentage  of 
cases  that  it  may  be  looked  upon  as  the  cause.  It  is  frequently  asso- 
ciated with  the  diplococcus  pneumoniae,  thus  giving  rise  to  a  mixed 
infection.  This  organism  is,  moreover,  an  etiological  factor  at  times 
in  other  inflammatory  diseases,  such  as  angina,  pleuritis,  pericarditis, 
endocarditis,  otitis,  suppurative  peritonitis,  etc. 

DIAGNOSIS. — The  bacillus  pneumoniae  resembles  very  closely  that 
of  ozena,  and  of  rhinoscleroma.  Moreover,  it  must  be  carefully 
distingushed  from  certain  varieties  of  the  colon  bacillus  (B.  aero- 
genes).  Glycerin  agar  plates,  grown  at  37°,  should  be  resorted  to  for 
the  detection  of  the  Friedlander  and  Friinkel  germs  in  suspected 
material. 

i  Make  gelatin  plates  and  cover-glass  preparations  from  the  lungs  and  blood  of  a 
young  rat  which  has  received  an  intrapleural  injection. 


DRAWINGS.  341 


Bacillus  Rhinoscleromatis,  Frisch  (1882). 

« 

KHINOSCLEROMA. 

ORIGIN. — In  the  tumors  of  rhinoscleroma,  a  rather  rare 
disease  which  occurs  in  Austria  and  Italy.  Very  rare  in 
this  country.  These  growths  may  appear  on  the  mucous 
membrane  of  the  nose  or  throat. 

FORM. — Short,  thick  rods  with  rounded  ends  resembling 
the  Friedlander's  pneumobacillus  and  the  B.  ozaense;  may 
form  short  threads.  They  are  likewise  surrounded  by  a 
colorless  capsule.  These  capsules,  when  masses  of  cells 
are  present,  coalesce  to  form  the  so-called  cells  of  Mikulicz. 

MOTILITY. — It  has  no  motion. 

SPORULATION. — This  has  not  been  observed. 

ANILIN  DYES. — Stain  readily,  and  show  a  colorless  cap- 
sule. The  bacillus  is  not  stained  by  Gram's  method. 

GROWTH. — This  resembles  in  almost  every  respect 
that  of  the  Friedlander  bacillus.  The  colonies,  stab  and 
streak  cultures  agree  so  closely  as  to  be  scarcely  distin- 
guishable. 

OXYGEN  REQUIREMENTS. — It  is  a  facultative  anaerobe. 

TEMPERATURE. — It  grows  rapidly  at  the  ordinary  tem- 
perature. The  optimum  is  about  36-38°. 

BEHAVIOR  TO  GELATIN. — This  is  not  liquefied. 

AEROGENESIS.— Gas  is  produced  when  grown  on  potato 
at  a  high  temperature. 

PATHOGENESIS. — The  relation  of  this  bacillus  to  rhin- 
oscleroma has  not  been  positively  established.  It  has  less 
virulence  than  the  Friedlander  bacillus.  It  is  pathogenic 
for  mice  and  guinea-pigs.  Rabbits  are  less  susceptible. 
The  bacilli  appear  in  the  blood  in  small  numbers. 

DIAGNOSIS. — The  constant  presence  of  the  bacillus  and 
the  ease  with  which  it  can  be  isolated  from  the  diseased 
tissue  is  of  some  diagnostic  value.  As  indicated  above,  the 

bacteria  occur  usually  in  groups. 

342 


DRAWINGS.  343 


Vibrio  Cholerae  Asiaticse,  Koch  (1884). 

CHOLERA  SPIRILLUM,  COMMA  BACILLUS;    BACILLE  VIRGULE  (>r.). 

ORIGIN. — In  the  excreta  of  cholera  patients,  also  in  the  intesti- 
nal contents  after  death.  Pound  several  times  in  the  water  supply. 

FORM. — Usually  appears  as  a  short,  rather  thick  rod  with 
rounded,  narrowed  ends,  and  with  a  more  or  less  decided  bend  or 
twist.  It  may,  therefore,  vary  from  apparently  a  straight  rod  to  one 
bent  in  form  of  a  half  circle.  Usually  the  bend  is  such  as  to  re- 
semble a  comma,  hence  the  name  comma  bacillus.  When  two  cells 
remain  attached  the  elongated/'S"  form  results.  Grown  in  liquid 
media  under  unfavorable  conditions  it  may  form  long"  spirals.  The 
bent  rod  is  a  segment  of  a  spirillum  and  is  designated  as  a  vibrio 
(Fig.  11,  p.  46).  Peculiar  involution  forms  develop  in  old  cultures. 

MOTILITY.— It  is  actively  motile  and  usually  has  at  one  end  a 
single  flagellum  (Fig.  6  d,  p.  36).  Hanging-drop  cultures  (p.  286) 
should  be  developed  at  37° — motion,  spirals  and  involutions. 

SPORULATION. — No  resistant  form  known.     Arthrospores  (p.  48'. 

ANILIN  DYES. — Stain  slowly.  Carbolic  fuchsin  is  excellent.  It  is 
not  stained  by  Gram's  method. 

GROWTH. — Is  fairly  rapid  at  the  ordinary  temperature. 

Plates. — On  gelatin  plates  kept  at  22°,  characteristic  colonies  develop  in  15-20  hours. 
The  colonies  appear  as  small,  white  points,  which  gradually  reach  the  surface  and  produce 
a  rather  slow  liquefaction  so  that  funnel-shaped  depressions  form.  After  several  days  the 
plate  becomes  wholly  liquefied.  Under  the  microscope,  the  colonies  show  an  irregular, 
rough  border;  have  a  white  or  pale-yellow  color  and  the  contents  are  coarsely  granular,  as 
if  made  up  of  broken  glass.  A  faint  rosy  hue  surrounds  the  border.  On  agar  plates,  at  37" 
the  large  colonies  have  'a  peculiar,  bright,  grayish-brown,  transparent  appearance,  quite 
distinct  from  that  of  the  comnron  bacteria  present  in  water  and  in  feces. 

Stab  culture. — Growth  occurs,  in  gelatin,  along  the  entire  line  of  inoculation.  At  the 
surface  a  funnel-shaped  liquefaction  forms  with  an  air  space  above,  while  the  lower  part 
contains  the  subsided  growth.  The  lower  part  of  the  puncture  gradually  widens  by  lique- 
faction; growth  settles  to  bottom,  and  eventually  entire  contents  of  tube  are  liquefied. 

Streak  culture. — On  agar  it  forms  a  bright,  glistening,  whitish  growth.  Blood-serum 
is  slowly  liquefied.  On  potato,  kept  in  the  incubator,  it  forms  a  grayish  or  yellowish  brown, 
thin  and  rather  transparent  layer,  which  resembles  somewhat  that  of  the  glanders  bacillus. 
At  ordinary  temperature,  no  growth  unless  a  mixed  culture  is  used. 

Bouillon.— Growth  takes  place  rapidly,  especially  in  the  incubator,  and  a  scum  or  pel- 
licle forms  on  the  surface.  Cultures,  12-24  hours  old,  on  addition  of  sulphuric  acid  show  a 
reddish-violet  color — the  indol  reaction  1 — due  to  formation  of  indol  and  nitrous  acid. 

Milk.— It  grows  abundantly  in  sterile  milk,  without  much  change;  also  in  sterile  water. 

OXYGEN  REQUIREMENTS.— Artificial  cultures  require  oxygen. 

TEMPERATURE.— Grows  at  15°-42°.     Optimum  at  37°.     Killed  at  50°. 

BEHAVIOR  TO  GELATIN.— Liquefies  slowly;    especially  old  cultures. 

IMMUNITY.— Subcutaneous  or  intra-peritoneal  injections  of  the 
dead  or  living-  vibrio  yield  an  anti-infectious  serum;  injections  of  the 
soluble  toxin  yield  an  antitoxic  serum.  The  cell  contents  of  the  cholera 
vibrios  immunize.  Pf  eiffer's  reaction  with  the  serum  of  conv'ales- 
cents,  or  that  of  immunized  animals  or  man  (Chap.  XIV). 

PATHOGEN ESIS. —Intravenous  injection  into  rabbits  kills  rapidly. 
In  guinea-pigs  intra-duodenal  injections  or  introduction  of  cultures 
into  the  previously  alkalized  stomach  produce  death  with  choleraic 
effects  (p.  272).  The  intraperitoneal  injection  of  agar  culture  is  ex- 
tremely fatal  to  guinea-pigs,— rapid  fall  of  temperature.  Subcutane- 
ous injection  of  pigeons  is  not  fatal— distinction  from  V.  Metchnikovi. 
Ingestion  of  cultures  produces  typical  cholera  in  man.  Feeding  of 
cultures  to  new-born  guinea-pigs  and  rabbits  is  usually  fatal  (p.  272). 

INFECTION.— Takes  place  along  the  alimentary  canal,  through  the 
water,  food,  contact  with  freshly  soiled  matter,  etc.  The  organism 
grows  in  the  intestines  and  the  soluble  poisons  which  are  elaborated 
there  induce  the  characteristic  symptoms  of  intoxication. 

DIAGNOSIS. — See  p.  346. 

i  Dissolve  i  g;  of  pepton  and  o.$  g.  of  NaCl  in  100  c.c.  of  ordinary  tap-water  (Dun- 
ham's solution).  Fill  into  tubes  and  sterilize  by  steam.  Then  inoculate  with  the  cholera 
and  other  vibrios,  and  place  in  the  incubator  over  night.  The  next  day  add  to  each  culture 
1-2  drops  of  sulphuric  acid  and  note  the  pink  color— the  indol  reaction  ' 

344 


DRAWINGS. 


Vibrio  Deneke,  Deneke  (1885). 
SPIRILLUM  TYROGENUM;  SPIRILLUM  OF  DENEKE;  CHEESE  SPIRILLUM » 

ORIGIN. — From  old  cheese. 

FORM.— Short,  slightly  bent  rods;  may  form  spirals.  Resem- 
bles somewhat  the  cholera  vibrio  but  it  is  smaller  and  the  comma-form 
is  less  pronounced. 

MOTILITY. — Very  motile.     Single  flagellum. 
SPORULATION. — Not  known. 
ANILIN  DYES.— Stain  readily. 

GROWTH.— Is  quite  rapid,  but  less  than  that  of  the  Finkler-Prior 
vibrio.  On  some  gelatin  media  it  liquefies  very  slowly  or  not  at  all. 
It  tends  to  die  out  rapidly. 

Plates.— The  colonies  which  develop  on  gelatin  plates  are  yellowish,  liquefy  and  the 
plate  as  a  whole  may  resemble  somewhat  that  of  the  cholera  vibrio,  but  the  liquefaction  is? 
more  rapid.  Under  the  microscope  they  are  seen  to  be  circular,  sharply  bordered  and 
coarsely  granular,  the  center  is  yellowish-green  and  later  becomes  dark  yellow. 

Stab  culture.— Growth  takes  place  in  gelatin  tubes  along  the  entire  line  of  inocula- 
tion; funnel-shaped  liquefaction.  The  mass  of  bacteria  settles  to  the  bottom  while  on  the 
surface  a  yellowish  scum  or  layer  forms. 

Streak  culture.— On  agar  a  thin  yellowish  growth  develops  along  the  line  of  inocula- 
tion. On  potato,  in  the  incubator,  it  forms  a  delicate,  yellowish  covering  which  frequently 
contains  well-formed  spirals.  Frequently,  no  growth.  On  blood-serum,  it  soon  produces 
liquefaction. 

Bouillon  cultures  are  cloudy  but  form  no  scum.  They  do  not  give  the  indol  reaction 
unless  a  nitrite  is  added. 

Milk  is  slowly  coagulated. 

OXYGEN  REQUIREMENTS. — Is  a  facultative  anaerobe. 
TEMPERATURE. — Grows  at  ordinary  temperature,  also  at  37°. 

BEHAVIOR  TO  GELATIN.— Liquefies  more  rapidly  than  the  cholera 
vibrio,  but  less  than  that  of  Finkler-Prior.  The  power  of  liquefac- 
tion is  subject  to  very  great  variation.  On  prolonged  cultivation  it 
may  almost  disappear— attenuation. 

AEROGENESIS. — Not  observed,  even  on  glucose  media. 

PATHOGENESIS. — It  is  less  pathogenic  to  guinea-pigs  than  the. 
Finkler-Prior  vibrio.  It  is  to  be  considered  as  a  saprophyte. 

DIAGNOSIS  OF  ASIATIC  CHOLERA. — When  making  microscopic  exam- 
inations of  intestinal  contents  it  should  be  remembered  that  comma 
bacilli  and  spirals,  which  are  derived  from  the  normal  mouth,  may  be 
present.  A  positive  recognition  of  the  cholera  vibrio  can  only  be 
effected  by  cultural  methods  such  as  are  given  in  Chapter  XIII.  This 
procedure  should  be  employed  not  only  to  effect  a  diagnosis,  but  also 
to  ascertain  and  confirm  the  absence  of  the  vibrio  from  the  intes- 
tines of  convalescents.  Although  they  usually  disappear  in  about  a 
week  they  may,  however,  persist  for  4  or  even  7  weeks,  in  which  case 
precaution  must  be  taken  to  prevent  the  spread  of  the  infection. 
Again,  in  cholera  times  apparently  healthy  persons  may  contain  the 
comma  bacillus.  In  such  cases  active  immunity,  or  unfavorable 
microbic  associations  may  prevent  development  of  this  organism. 

346 


DRAWINGS. 


Vibrio  Finkler-Prior,  Finkler  and  Prior  (1884). 

VIBRIO  PROTEUS,    FINKLER-PRIOR'S   SPIRILLUM. 

ORIGIN. — In  the  dejections  of  cholera  nostras  which  had  been 
kept  fourteen  days.  It  is  not  the  cause  of  cholera  nostras.  Appar- 
ently the  same  organism  has  been  found  in  a  tooth  cavity  (V.  Miller), 
and  also  in  normal  intestines. 

FORM. — It  resembles  the  cholera  vibrio  but  is  longer  and  thicker: 
occasionally  forms  short  spirals.  Marked  tendency  to  involution 
forms,  hence  the  name  V.  proteus.  These  develop  especially  in 
gelatin  which  contains  glucose  or  glycerin. 

MOTILITY. — It  is  actively  motile  and  has  a  single  flagellum  at  one 
end. 

SPORULATION. — This  has  not  been  observed. 
ANILIN  DYES. — Stain  readily. 

GROWTH. — Is  much  more  rapid  than  that  of  the  cholera  vibrio, 
from  which  it  can  be  easily  distinguished. 

Plates. — The  colonies  develop  rapidly  on  gelatin  plates  and  produce  extensive  circu- 
lar liquefactions  which  are  diffusely  clouded.  Under  the  microscope  they  appear  as  yellow- 
ish brown,  finely  granular  masses,  the  contents  of  which  can  be  seen  to  possess  marked 
motion.  The  edge  is  dark  and  is  beset  with  short  delicate  fibrils.  Concentric  rings  may 
be  present. 

Stab  culture. — In  gelatin  tubes,  rapid  growth  and  liquefaction  along  the  entire  line  of 
inoculation — the  so-called  stocking-shaped  liquefaction.  In  a  few  days  the  entire  contents 
are  liquefied,  and  a  thin  film  usually  forms  on  the  surface. 

Streak  culture. — On  agar  it  forms  a  thick,  moist,  slimy,  spreading  growth.  On  Potato 
growth  occurs  rapidly,  even  at  ordinary  temperature,  forming  a  grayish-yellow,  slimy,  glis- 
tening, spreading  mass.  Distinction  from  the  comma  bacillus  which  does  not  grow  on 
potato  at  the  room  temperature.  On  blood-serum,  rapid  development  with  liquefaction. 

Milk.— It  can  grow  in  milk,  which  it  coagulates;  in  water  it  soon  dies  out.  Abundant 
growth  in  bouillon.  No  indol  unless  nitrite  is  added. 

OXYGEN  REQUIREMENTS.— It  is  a  facultative  anaerobe. 

TEMPERATURE. — It  grows  well  at  ordinary  temperature;  likewise, 
in  the  incubator. 

BEHAVIOR  TO  GELATIN. — This  is  liquefied  rapidly  and  extensively. 
AEROGENESIS. — No  gas  observed.     Disagreeable  odor  evolved. 

PATHOGENESIS.— As  a  rule,  it  is  fatal  to  guinea-pigs  if  the  bouillon 
cultures  are  introduced  into  the  previously  alkalinized  stomach.  The 
intestines  are  pale  and  contain  watery  contents.  Intra-peritoneal 

injections  are  less  fatal  than  those  of  the  comma  bacillus. 

348 


DRAWINGS. 


Vibro  Metchnikovi,  Gamaleia  (1888). 

SPIRILLUM  METCHNIKOFF. 

ORIGIN.—  From  the  intestinal  contents,  blood  and  organs  of  chick- 
•ens  afflicted  with  a  disease  resembling  chicken  cholera.  The  disease 
exists  in  Russia  during"  the  summer  months  and  is  due  to  this  organ- 
ism. Also  found  in  the  Spree  at  Berlin  (Vibrio  Nordhafen). 

FORM.  —  Occurs  as  a  bent  rod  which  bears  a  marked  resemblance 
to  the  vibrio  of  Asiatic  cholera,  although  it  is  somewhat  shorter  and 
thicker  and  more  decidedly  bent.  In  the  animal  body  it  is  very  short, 
almost  a  coccus.  Typical  spirals  may  form  in  old  cultures. 

MOTIIJTY.—  Very  actively  motile.  It  possesses  a  long,  slender 
whip  at  one  end. 

SPORULATION.  —  Has  not  been  observed.  It  is  readily  destroyed, 
like  the  cholera  vibrio,  by  heat,  desiccation,  acids,  etc. 

ANILIN  DYES.  —  Stain  very  well,  especially  if  warmed.  Bi-polar 
stain  may  be  seen  if  the  dye  is  weak.  Gram's  method  is  not  applicable. 

GROWTH.—  In  the  hanging-drop  and  in  stained  preparations  this 
organism  can  scarcely  be  distinguished  from  the  cholera  vibrio.  The 
cultural  properties  show  some  differences,  and  especially  is  this  seen 
in  the  pathogenic  action  on  animals.  The  rapidity  of  growth  is 
greater  than  that  of  the  cholera  vibrio  and  less  than  that  of  the 
Finkler-Prior  vibrio. 

Plates.—  The  colonies  on  gelatin  plates  may  resemble  those  of  the  cholera  vibrio  and 
also  those  of  the  Finkler-Prior.  They  are  circular,  coarsely  granular,  and  yellowish.  Rapid 
liquefaction. 

Stab  culture.  —  In  gelatin  tubes  the  growth  resembles  that  of  the  cholera  vibrio,  which 
is  about  twice  as  old.  Eventually  they  are  both  alike. 

Streak  culture.  —  On  agar  the  growth  resembles  that  of  the  cholera  vibrio.  It  is  fairly 
thick  and  yellowish.  On  potato  in  the  incubator  it  forms  a  moderate  yellowish  brown 
covering. 

Bouillon.—  Abundant,  diffuse  growth  and  a  whitish  scum  forms.  It  gives  a  more 
pronounced  indol  reaction  than  does  the  comma  bacillus. 

.-^\?>  slowly  coagulated  and  acid  products  form. 


OXYGEN  REQUIREMENTS.  —  Same  as  cholera  vibrio. 
TEMPERATURE.  —  Same  as  cholera  vibrio. 

BEHAVIOR  TO  GKLATIN.  —  Grows  and  liquefies  more  rapidly  than 
does  the  comma  bacillus. 

AEROGENESIS.  —  No  gas  is  produced  in  glucose  media. 

IMMUNITY.—  Is  conferred  on  pigeons  and  guinea-pigs  by  injection 
of  sterilized  cultures.  Such  animals  are  not  immune  to  the  comma 
bacillus. 

PATHOGENESIS.—  It  is  very  infectious  for  guinea-pigs,  pigeons,  and 
•chicken;  rabbits  are  also  susceptible.  Guinea-pigs  and  pigeons,  after 
subcutaneous  injection  of  minute  amounts,  die  within  24  hours—  dis- 
tinction from  the  cholera  vibrio,  which  is  not  as  pathogenic  and  does 
not  induce  a  septicemia  as  in  the  above  case.  The  vibrios  are  abun- 
dant in  the  blood,  in  the  internal  organs,  and  in  the  serous  fluid 
which  permeates  the  muscles.  Old  sterilized  cultures  are  very 
toxic  and  cause  a  rapid  fall  in  temperature. 

350 


DRAWINGS.  351 


Bacillus  Coli  Communis,  Escherich  (1885). 

» 

BACTERIUM  COLI  COMMUNE;    THE  COLON  BACILLUS;   EMMERICH'S 

BACILLUS;  B.  NEAPOLITANUS. 

ORIGIN.— It  is  very  common  and  constant  in  the  intestinal  con- 
tents of  man  and  animals,  especially  in  the  colon;  occurs  in  the  dis- 
charges of  healthy  infants,  also  in  summer  diarrhea.  It  is  frequently 
present,  accompanying1  the  comma  bacillus,  in  the  discharges  of 
Asiatic  cholera,  and  in  later  stages  may  be  the  only  organism  pres- 
ent. Not  infrequently  it  is  present  in  pus  (B.  pyogenes  foetidus).  It 
is  apparently  the  most  frequent  cause  of  appendicitis.  In  many 
respects  it  resembles  the  typhoid  fever  bacillus. 

FORM.— Short,  narrow  rods;  may  vary  in  length  from  oval  or 
coccus-like  forms  to  rods  4-6  times  as  long  as  wide.  Usually  grows  in 
pairs;  occasionally  in  short  threads. 

MOTILITY. — Exceedingly  variable,  depending  upon  the  tempera- 
ture, age  and  the  medium.  Diffuse  flagella  and  giant-whips  present. 

SPORULATION.— Not  observed. 

ANILIN  DYES.— It  stains  readily,  but  not  by  Gram's  method.  Bi- 
polar stain  frequently  met  with;  likewise  plasmolytic  changes,  as  in 
potato  cultures. 

GROWTH. — Is  more  rapid  than  that  of  the  typhoid  bacillus. 

Plates. — On  gelatin  plates  the  surface  colonies  are  flat,  spreading,  aniso-diametricr 
and  have  a  dull-white  color.  The  border  is  irregular  ,and  markings  are  present  in  the  outer 
zone.  The  deep  colonies  are  yellowish,  round  or  oval;  frequently  divided,  forming  lobu- 
lated  masses.  The  deep  circular  colonies  usually  show  a  yellow  granular  center  which  is 
surrounded  by  a  colorless,  homogeneous  ring  (dish  appearance).  No  liquefaction.  Strong 
amine  and  indol  order.  Moreover,  the  gelatin  owing  to  the  ammoniacal  reaction,  deposits 
a  cloudy  precipitate  between  the  colonies,  and  along  any  scratches  that  may  be  on  the  glass- 
plate.  These  characteristics  are  not  given  by  the  typhoid  bacillus. 

Stab  culture. — Rather  energetic  growth  along  the  line  of  inoculation,  while  on  the  sur- 
face it  spreads  as  a  white  film  with  wavy  border. 

Streak  culture. — On  agar,  it  forms  a  moist,  wttite,  spreading  growth;  old  cultures 
may  show  needle-shaped  crystals.  On  potato,  it  forms  an  abundant,  yellowish,  moist, 
slowly  spreading  growth. 

Milk. — Is  coagulated  in  one  or  two  days,  though  at  times  a  week  or  more  may  be 
necessary. 

Uschinsky^s  fluid  gives  a  good  growth;  likewise  a  solution  of  ammonium  chloride  and 
glycerin — distinction  from  the  typhoid  bacillus. 

Bouillon  becomes  very  clouded.  The  sediment  is  heavy  and  a  thick  ring  may  adhere 
to  the  glass  at  the  surface  of  the  liquid.  At  times  a  broken  pellicle  may  form.  Indol  reac- 
tion is  marked. 

Hiss1  and  Stoddart's  media  yield  diffuse  growths  according  to  the  motility  of  the 
species. 

OXYGEN  REQUIREMENTS. — Is  a  facultative  anaerobe. 

TEMPERATURE.— Grows  well  at  ordinary,  temperature.  Its  opti- 
mum is  about  37°. 

BEHAVIOR  TO  GELATIN. — Does  not  liquefy. 

AEROGENESIS. — Carbonic  acid  and  hydrogen  is  produced  in  abund- 
ance when  glucose  is  present.  Unlike  the  typhoid  bacillus  it  can 
form  acid  and  gas  in  lactose  media. 

PATHOGENESIS.— Guinea-pigs  are  very  susceptible;  rabbits  less  so; 
mice  insusceptible.  Small  quantities  injected  intravenously  or  into 
the  abdominal  cavity  produce  diarrhea,  collapse  and  death  in  1-3 
days.  The  small  intestine  is  hyperemic.  more  or  less  intensely  in- 
flamed; serous  exudates  may  be  present.  The  bacilli  are  abundant  in 
the  blood  and  organs  and  on  the  peritoneum.  Subcutaneous  injection 
is  usually  non-fatal  and  produces  only  a  local  abscess. 

DIAGNOSIS. — See  p.  364. 

352 


DRAWINGS.  353 


23 


Bacillus  Typhosus,  Eberth,  Koch  (1880). 

BACILLUS  OF  TYPHOID  FEVER;   KOCH-EBERTH'S  BACILLUS. 

ORIGIN. — First  obtained  from  the  spleen  and  lymphatic  glands  of 
typhoid  fever  cadavers;  present  in  the  blood,  though  in  small  num- 
bers: also  in  tlje  feces  and  urine  of  typhoid  patients. 

'FORM. — Good  sized  rods,  3-5  times  as  long-  as  wide,  with  rounded 
ends.  The  length  varies  greatly  with  the  nature  of  the  medium  on 
which  it  grows.  Thus,  on  agar  it  appears  as  very  short  rods,  while  on 
the  potato  it  may  grow  into  long  threads.  Involution  forms. 

MOTILITY. — Is  very  actively  motile:  on  prolonged  artificial  cul- 
ture it  may  show  little  or  no  motion.  It  has  numerous  lateral  whips. 
Excellent  giant-whips. 

SPORULATION. — Terminal,  round  or  oval  bodies  are  found  in  potato 
and  agar  cultures,  which  are  grown  in  the  incubator  for  several  days. 
They  do  not  double  stain  and  the  bacilli  containing  these  are  very 
susceptible  to  heat.  They  are  not  therefore  true  spores,  but  rather 
little  masses  of  condensed  protoplasm  (see  p.  27).  The  bacilli  are 
very  resistant  to  desiccation  and  may  retain  their  vitality  for  months. 

ANILIN  DYES.— It  does  not  stain  as  well  with  ordinary  anilin  dyes 
as  do  most  bacteria:  carbolic  fuchsin  stains  excellently.  It  does  not 
stain  by  Gram's  method.  Excellent  bi-polar  stain  when  grown  on 
potato;  no  plasmolysis  (Migula). 

GROWTH.  —Is  less  rapid,  but  in  its  cultural  properties  it  greatly 
resembles  the  preceding  organism.  Slow  at  16-18°. 

Plates. — The  deep  colonies  on  gelatin  plates  are  small,  round,  or  oval,  sharply  bor- 
dered, finely  granular  and  yellowish.  They  may  show  a  central  dark  portion  or  ring.  Fre- 
quently, a  protuberance  or  swelling  will  be  seen  on  the  border  of  the  colony.  At  times  deli- 
cate fibrils  may  surround  the  border  (see  Eisner's  medium).  The  surface  colonies  (crater- 
like)  spread  freely  as  an  almost  transparent  film,  which  has  an  irregular,  wavy  border,  and 
is  delicately  marked  with  branching  lines.  No  liquefaction.  For  appearance  of  colonies  on 
Eisner's  or  On  Stoddart's  medium,  see  Chapter  XIV.: 

Stab  culture.— Abundant  growth  along  the  entire  line  of  inoculation,  and  especially 
so  on  the  surface  where  it  spreads  as  a  thin,  grayish  white  covering.  Gelatin  eventually 
becomes  cloudy,  due  to  the  production  of  acids.  See  also  stab  culture  in  Hiss'  medium. 

Streak  culture, — On  agar  and  on  blood-serum  it  forms  a  moist,  white  growth,  without 
any  special  characteristics.  On  potato,  as  a  rule,  the  growth  is  very  characteristic.  It 
covers  the  surface  as  a  moist,  invisible  layer— distinction  from  preceding  organism.  On 
alkaline  potato,  the  growth  is  yellowish  and  no  longer  characteristic.  For  litmus  lactose 
agar  see  p.  241. 

Bouillon. — Slight  diffuse  -cloudiness,  much  less  than  with  the  colon  bacillus.  Very 
little  deposit  and  scarcely  any  ring  or  film.  Remains  clouded  for  a  long  time.  Unlike  the 
colon  bacillus,  it  will  not  grow  in  bouillon  which  contains  20  c.c.  of  N  HC1  or  50  c.c.  of  N 
NaOH  per  liter.  No  indol  is  produced.  No  growth  in  Uschinsky's  medium. 

Milk. — Is  not  coagulated.    No  gas  in  glucose  media,  nor  acid  in  lactose  media. 

OXYGEN  REQUIREMENTS.— It  is  a  facultative  anaerobe. 

TEMPERATURE. — Grows  well  at  ordinary  temperature.  Optimum 
at  37°.  Exposure  of  a  few  minutes  to  moist  heat  of  60°  kills. 

AEROGENESIS.— No  acid  or  gas  production  on  lactose  media. 

BEHAVIOR  TO  GELATIN. — Does  not  liquefy. 

IMMUNITY. — As  in  cholera,  injections  of  dead  or  living  cultures 
yield  an  anti-infectious  serum,  whereas  injections  of  the  toxin  yield 
an  antitoxic  serum.  The.serum  in  the  former  case  will  give  Pf  eiffer's 
reaction  with  the  Eberth*  but  not  with  the  colon  bacillus. 

PATHOGENEsrs. — Intravenous  injections  usually  kill  rabbits.  It  is 
usually  fatal  to  guinea-pigs,  when  introduced  into  the  previously 
alkalized  stomach,  or  when  injected  into  the  duodenum  or  into  the 
peritoneal  cavity.  Subcutaneous  injections  are  also  fatal  to  guinea- 
pigs  -distinction  from  the  colon  bacillus.  The  same  method  of  infec- 
tion will  produce  abscesses  in  rabbits  and  dogs.  At  times  it  produces 
abscesses  in  man.  Cultures  killed  with  chloroform,  or  by  heating  at 
54°  for  one  hour  are  fatal  to  guinea-pigs  in  a  dose  of  3-4  mg.  per  100  g. 
body-weight. 

INFECTION,  DIAGNOSIS. — See  p.  382. 

354 


DRAWINGS.  355 


Bacillus  Icteroides,  Sanarelli  (1897). 


ORIGIN.  —  In  the  blood  and  organs  in  yellow  fever.  Although 
present  in  small  numbers  and  isolated  only  in  7  out  of  13  cases,  it  is 
regarded  by  Sanarelli  as  the  cause  of  the  disease. 

FORM.  —  Small,  very  slender  rods;  two  to  three  times  as  long  as 
wide.  It  is  usually  single  or  in  pairs;  short  threads  are  occasionally 
found  in  bouillon.  In  the  body  and  on  agar  it  is  almost  coccus-like  in 
appearance,  whereas  in  bouillon  large  rods  are  met  with.  Bright 
polar  bodies. 

MOTILITY.  —  Extremely  motile,  especially  in  the  water  of  conden- 
sation on  agar.  It  bears  4-8  long  whips.  Splendid  giant-whips  (Novy). 

SPORULATION.  —  Not  observed  in  cultures  of  the  typical  bacillus. 

ANILIN  DYES.—  Stain  readily.  Gram's  method  is  not  applicable. 
At  times,  a  bi-polar  stain  may  be  obtained. 

GROWTH.  —  Is  rapid  and  on  some  media  very  characteristic.  In 
general,  it  resembles  quite  closely  that  of  the  typhoid  bacillus. 
Rapidity  of  growth  is  like  that  of  B.  typhosus  and  less  than  that  of 
the  colon  bacillus.  No  growth  on  distinctly  acid  media. 


Plates.  —  The  colonies  on  gelatin  plates  are  extremely  characteristic.    To  obtain  typi- 
c " 

p. 


cal  colonies  it  is  necessary  to  maintain  a  constant  temperature  of  16-18"  (See  Fig.  33, 
p.  179);  moreover,  the  gelatin  should  not  be  too  hard  (10  per  cent.  9r  less)  and  should  be 
alkalin  (ep.  157).  Under  proper  conditions,  as  indicated,  characteristic  colonies  can  be 


obtained  from  cultures  which  have  been  kept  in  the  laboratory  for  several  years  (Novy). 
Otherwise,  atypical  colonies  will  result. 

The  deep  colonies  are  perfectly  circular  and  sharp  bordered.  At  first  they  are  light 
yellow,  almost  homogeneous,  wax-like  in  appearance.  Later  they  become  dark  or  perfectly 
black.  They  may  show  at  the  center  slight,  irregular  radiating  lines.  Atypical  colonies  are 
those  which  do  not  become  black;  those  which  are  lobular  or  are  surrounded  by  a  fringe  of 
fibrils.  The  latter  two  forms  are  rather  rare.  The  former  develop  on  acid  gelatin. 

The  surface  colony  appears  to  the  eye  like  a  droplet  of  boiled  starch  or  mucus.  It  is 
thick  and  convex.  It"  may  be  perfectly  circular,  but  frequently  will  show  a  distinct 
kidney-shape.  An  opaque,  yellowish  white  nucleus  can,  as  a  rule,  be  seen  at  or  near  the 
center;  or,  in  the  case  of  the  kidney-shaped  colony  at  the  hilum.  Under  the  microscope  the 
border  appears  colorless,  or  with  a  central,  light-brown  tinge.  It  is  finely  granular  and  the 
border  is  sharp  perfectly  smooth.  The  nucleus  is  opaque  and  may  be  round,  but  more 
often  is  hat-shaped,  and  has  even  been  compared  to  Saturn  with  its  rings.  In  the  kidney- 
shaped  colony  the  crown  of  the  hat  projects  into,  or  is  turned  toward  the  hilum.  On  hard  or 
acid  gelatin  only  atypical  colonies  form.  These  are  small,  raised  points  usually  without  the 
nucleus,  and  the  kidney  shape  is  absent. 

Isolated  colonies  on  inclined  agar  (p.  239),  developed  at  39°,  are  thin,  flat,  grayish  and 
circular.  If  at  the  end  of  24  hours  the  tubes  are  placed  at  16°,  growth  at  the  edge  of  each 
colony  will  continue,  but  it  will  take  on  a  thick,  slimy  character.  In  two  or  three  days  the 
colonies  present  a  peculiar  appearance—  a  thick,  opalescent,  slimy  ring  surrounds  a  flat, 
thin,  transparent  area.  When  several  colonies  are  close  together  the  slimy  growths  may 
coalesce  and  give  rise  to  rivulets.  Eventually,  the  outer  slimy  border  becomes  transparent, 
and  the  original  colony  is  seen  as  an  opaque  body  imbedded  in  it. 

Stab  culture.—  It  grows  slowly  along  the  line  of  inoculation  and,  unless  kept  at  about 
25°,  does  not  tend  to  spread  on  the  surface. 

Streak  culture.  —  At  39°  on  agar,  like  the  B.  typhosus  it  forms  a  thin,  transparent 
growth.  At  16  it.forms  a  thick,  moist  slimy  growth,  which  is  homogeneous  and  mucous- 
Rke  in  appearance,  and  is  not  unlike  that  of  Friedlander's  bacillus.  On  potato,  it  yields  a 
moist,  colorless,  invisible  growth.  On  blood-serum  a  scarcely  visible,  transparent  film  forms. 

Bouillon.—  No  ring  or  pellicle  as  a  rule,  forms  on  the  surface.  The  liquid  is  clouded 
and  remains  so  for  some  time.  Very  little  sediment  in  the  tube.  Involution  forms. 

Milk.  —  Is  not  coagulated.    Indol  reaction  is  not  given,  or  but  very  faintly. 

On  glucose  media  it  produces  gas,  also  an  acid  reaction.  No  growth  in  Uschinsky;s 
or  in  Eisner's  medium.  On  Stoddart's  medium  it  forms  a  thin,  transparent,  rapidly  spread- 
ing film.  Similar  diffusion  in  Hiss's  tube  medium. 

OXYGEN  REQUIREMENTS.—  It  is  a  facultative  anaerobe. 

TEMPERATURE.—  Has  a  marked  effect  upon  the  character  of  the 
growth.  Grows  at  14-40°.  The  optimum  is  at  about  37°. 

BEHAVIOR  TO  GELATIN.—  It  does  not  liquefy. 

ATTENUATION.  —  It  is  not  affected  by  freezing  for  some  days. 

IMMUNITY.  —  An  antitoxic  serum  has  not  been  obtained.  Normal 
blood  agglutinates. 

PATHOGENESIS,  DIAGNOSIS.  —  See  p.  382. 

356 


DRAWINGS.  357 


Bacillus  Pestis  Bubonicse,  Yersin,  Kitasato  (1894). 

BLACK  OR  BUBONIC  PLAGUE;  BLACK  QEATH;  PEST. 

ORIGIN. — In  the  enlarged  or  suppurating-  glands  or  buboes;  in 
sputum  in  the  pneumonic  form  of  the  disease.  Also  present  in  the 
urine  and  f  eces.  In  severe  cases  may  be  present  in  small  numbers  in 
the  blood. 

FORM.— A  short,  thick  bacillus  with  rounded  ends  (cocco-bacillux). 
At  times,  may  be  almost  a  coccus  and  may  form  short  chains,  of  6-8 
cells.  It  is  subject  to  great  variation  in  form,  and  not  infrequently 
is  surrounded  by  capsules.  Involution  forms  are  frequent,  especially 
in  the  presence  of  mercury,  salt  and  other  antiseptics. 

MOTILITY.— It  is  said  to  be  non-motile.  May  have  1  or  2  flagella 
(Gordon).  Browniaji  motion  is  marked. 

SPORULATION.— No  spores  observed.  It  is  a  relatively  weak  organ- 
ism; a  few  minutes  at  58°  kills. 

ANILIN  DYES. — It  stains  readily;  Gram's  method  is  not  applica- 
ble. At  times,  a  bi-polar  stain  may  be  observed  and  in  the  case  of 
short  threads  the  resulting  appearance  is  not  unlike  that  of  the 
"beaded"  tubercle  bacillus.  Abnormal  forms  do  not  stain  well. 

GROWTH  . — Is  slow  and  not  abundant.  The  cultures  die  out  readily. 
They  are  usually  slimy. 

Plates.— The  colonies  on  cigar  are  small,  transparent  and  white  with  iridescent 
edges.  On  gelatin  the  colonies  are  very  small,  circular  and  sharply  bordered;  eventually 
they  become  brown  and  coarsely  granular. 

Stab  culture. — Shows  a  very  slight  white  growth  along  the  line  of  inoculation,  and 
spreads -but  little  on  the  surface.  At  times,  minute  branches  extend  into  gelatin  from  the 
puncture,  the  resulting  appearance  resembles  somewhat  an  anthrax  stab  culture. 

Streak  culture. — On  agarii  forms  a  rather  thin,  whitish,  slimy  growth  which  usually 
is  made  up  of  discrete  colonies.  The  old  cultures  show  large  coccus-,  or  torula-like  forms, 
and  but  very  few  typical  ovals  or  short  rods.  Glycerin  agar  yields  a  better  growth.  It 
grows  well  oh  serum ;  poorly  on  potato. 

Bouillon. — In  this  the  growth  is  very  characteristic  and  resembles  that  of  the  strep- 
tococci. The  liquid  usually  remains  clear  while  the  granular  or  slightly  flocculent,  moderate 
growth  develops  on  the  walls  and  bottom  of  the  tube.  Old  cultures  show  a  ring  or  "col- 
laret" on  the  surface  of  the  liquid.  A  bouillon  with  2  per  cent,  each  of  pepton  and 
glycerin  furnishes  the  best  medium.  On  staining,  various  bizarre  forms  are  met  with.  Short 
threads  of  6-8-10  cells  like  streptococci  are  present.  Good  rods  may  be  present  but  the 
size  of  the  cells  varies  greatly. 

Milk, — Is  not  coagulated.  Acid  is  produced  in  bouillon.  An  indol  reaction  results 
on  the  addition  of  a  nitrite. 

OXYGEN  REQUIREMENTS.— It  can  grow  as  a  facultative  anaerobe, 
but  only  in  gelatin. 

TEMPERATURE. — It  grows  well  at  the  ordinary  room  temperature. 
Prolonged  exposure  to  37°  is  unfavorable.  Cold  has  no  effect. 

BEHAVIOR  TO  GELATIN.— It  does  not  liquefy. 

AEROGENESIS. — Not  observed. 

ATTENUATION. — Takes  place  readily.  Cultures  from  convalescents 
may  be  non-virulent,  Other  growths  rapidly  lose  their  virulence  on 
artificial  culture.  By  successive  passage  through  animals  or  by*  the 
collodium  sac  method  (Chapter  XIV),  the  virulence  may  be  greatly 
increased.  A  culture  of  maximum  virulence  obtained  by  passage 
through  mice  is  feeble  with  reference  to  rabbits,  and  vice  versa. 

IMMUNITY. — Can  be  obtained  by  means  of  agar  cultures.  The 
injections,  at  first,  are  made  with  cultures  heated  at  58°  for  1  hour. 
Subsequently,  living  cultures  are  injected  intravenously.  The  rabbit 
and  horse  can  be  thus  readily  immunized,  whereas  guinea-pigs  are 
difficult.  The  serum  of  the  immunized  horse  is  anti-infectious  and  to 
less  extent  antitoxic.  As  usually  tested  0.1  c.c.  of  the  serum  should 
protect  a  white  mouse  against  10  mg.  of  the  toxin  which  has  been 
precipitated  by  (NH4)2SO4.  2.5  mg.  of  this  toxin  will  kill  a  control 
mouse  in  about  12  hours.  Vaccination  by  repeated  injection  of  bouil- 
lon cultures,  heated  to  70°  (Haffkine). 

PATHOGENESIS,  INFECTION  AND  DIAGNOSIS. — See  p.  383. 

358 


DRAWINGS.  359 


Bacillus  Influenzae,  Pfeiffer  (1892). 
INFLUENZA;  LA  GRIPPE  (Fr.). 

ORIGIN.— In  the  greenish-yellow,  purulent  sputum  of  the  disease; 
present  in  masses  in  the  nasal  and  bronchial  secretions;  rarely  in  the 
blood.  In  the  early  stage  they  are  free,  whereas  later  on  they  are  in- 
cluded within  pus  cells. 

FORM. — Extremely  small,  rather  plump  rods;  usually  in  pairs,  very 
rarely  in  threads.  Involutions  may  be  present  in  old  cultures.  Branch- 
ing" forms  observed.  The  pseudo-influenza  bacillus  forms  decidedly 
larger  rods  which  have  a  marked  tendency  to  form  threads. 

MOTILITY. — It  has  no  motion. 

SPORULATION. — This  has  not  been  observed.  It  is  very  sensitive  to 
desiccation. 

ANILIN  DYES. — Stain  with  some  difficulty.  Loffler's  methylene  blue 
and  carbolic  fuchsin  are  useful.  Gram's  method  is  not  applicable.  At 
times,  a  bi-polar  stain  may  be  observed. 

GROWTH. — Is  slight  and  requires  a  special  medium  (see  below)  and 
the  body  temperature.  Moreover,  the  culture  should  be  transplanted 
every  3  or  4  days.  No  growth  on  ordinary  agar  or  serum.  It  is  favored 
by  association  with  staphylococci.  The  addition  of  defibrinated  blood 
to  agar  at  100°  yields  a  good  medium  (Voges). 

Plates.— The  colonies  on  agar-blood  appear  as  isolated,  minute,  glassy  drops  which, 
under  the  microscope,  appear  colorless  and  homogeneous.  Later  on,  the  middle  of  the  col- 
ony may  show  a  yellowish  or  brownish  color.  They  remain  discrete  and  are  usually  so 
small  that  a  lens  may  be  necessary  to  reveal  them. 

Stab  culture. — Very  slight  growth  along  the  puncture  in  hamatogen-agar. 

Streak  culture. — Isolated,  dew-like  colonies  on  agar-blood.  Vitality  may  persist  for 
2-3  weeks. 

Somllon.—'Blood  must  be  added  to  the  medium  which,  moreover,  must  be  shallow. 
Delicate  white  floccules  form.  The  cultures  retain  their  vitality  for  2-3  weeks.  In  sterile 
tap-water  it  dies  out  in  about  24  hours. 

OXYGEN  REQUIREMENTS. — It  appears  to  be  anobligative  aerobe. 

TEMPERATURE.— Optimum  at  37°.     May  grow  at  26  to  42°. 

ATTENUATION. — It  is  readily  destroyed  by  mere  desiccation,  and  is 
even  more  sensitive  in  this  respect  than  the  cholera  vibrio. 

IMMUNITY. — Has  not  been  established  in  guinea-pigs  even  after 
prolonged  treatment  with  living  and  dead  cultures. 

PATHOGENESIS. — The  effects  on  animals  are  not  very  characteris- 
tic. Dead  or  living  cultures  injected  intravenously  in  rabbits  produce 
fever,  weakness  and,  at  times,  death;  subcutaneously  they  induce  ab- 
scesses. Mice  and  guinea-pigs  are  less  susceptible.  Monkeys  react 
with  a  fever.  The  disease  is  primarily  one  of  the  respiratory  pass- 
ages from  whence  toxic  products  are  absorbed.  A  somewhat  similar 
organism,  Bacillus  conjunctivitidis,  is  found  in  a  catarrhal  eye  disease. 
The  latter  is  present  in  large  numbers  in  the  pus  cells  and  can  be 
cultivated  on  agar-blood  medium  (Diplo-bacillus  of  Weeks). 

DIAGNOSIS. — In  the  early  stage  a  microscopical  examination  will 
show  large  numbers  of  the  characteristic  rods.  Otherwise  it  is  neces- 
sary to  isolate  the  organism.  This  is  done  as  follows:  Agar  is  poured 
into  Petri  dishes  and  allowed  to  solidify.  Some  human  or  rabbit 
blood  (Chap.  XIV)  is  then  spread  over  the  surface  of  the  plate.  Fin- 
ally the  material  itself,  from  a  fresh  acute  case,  is  rubbed  up  in  ster- 
ile bouillon  and  is  streaked  over  the  plates,  thus  prepared,  which  are 
then  set  aside  at  37°.  Obviously  inclined  agar  tubes  may  be  also  em- 
ployed. The  material  should  also  be  streaked  over  the  surface  of 
plain  agar  in  which  case  failure  to  obtain  growth  would  confirm  the 
nature  of  that  which  developed  on  agar-blood. 

360 


DRAWINGS.  361 


Bacillus  Pyocyaneus,  Gessard  (1882). 

BACILLUS  OF  GREEN  OR  BLUE  PUS. 

ORIGIN.— In  green  pus.  The  color  forms  on  exposure  to  air.  Sev- 
eral varieties  have  been  described.  It  is  very  widely  distributed  in 
nature.  It  has  been  found  on  the  skin  and  in  the  mouth,  nose,  stoni- 
ach  and  intestines.  Not  infrequently,  it  is  found  in  the  lungs,  blood 
and  internal  organs  of  men  and  animals;  in  dust,  soil  and  in  water. 
In  many  respects  it  resembles  the  liquefying1,  fluorescing  bacillus. 

FORM.— Small  narrow  rod  like  that  of  blue  milk.  At  times 
almost  a  coccus.  The  ends  are  rounded.  It  is  usually  single,  may 
form  short  threads  of  4-6  cells,  or  even  longer,  spiral-like  filaments. 

MOTILITY. — Actively  motile.     Single  polar  whip. 

SPORULATION. — Has  not  been  observed. 

ANILIN  DYES.— It  stains  easily;  also  by  Gram's  method. 

GROWTH. — Is  rapid  and  abundant.  Oxygen  is  necessary  to  the 
formation  of  pigment. 

Plates.— On  gelatin  plates  a  green  fluorescing  pigment  develops  quite  early.  The 
surface  colonies  at  first  tend  to  spread;  then  produce  funnel-shaped  liquefactions.  The  deep 
colonies  appear  as  round,  coarsely  granular  masses  with  serrated  borders.  They  are  yel- 
lowish and  may  show  radial  markings. 

Stab  culture.— In.  gelatin  tubes  funnel-shaped  or  cylindrical  liquefaction  results.  The 
upper  layer  is  at  first  green  but  later  the  entire  contents  are  colored.  In  very  old  cultures 
the  color  changes  to  a  brownish  black.  A  scum  forms  on  the  surface. 

Streak  culture. — On  agar  a  moist,  slimy,  yellowish  growth  develops  and  the  medium 
itself  becomes  bright  green.  When  very  old  the  agar  becomes  dark  colored  and  the  growth 
has  a  peculiar  scaly,  metallic  appearance.  On  potato  a  yellowish  green  or  brownish  slimy 
growth  forms. 

Milk.— Grayish  yellow  spots  form  on  the  surface;  the  casein  is  precipitated  (rennet 
action);  subsequently  it  is  peptomzed  with  production  of  ammonia. 

Bouillon. — Becomes  very  cloudy  and  a  heavy  deposit  forms.  A  thick  white  scum 
forms  on  the  surface.  The  upper  layer  of  the  liquid  is  greenish.  Indol  is  produced. 

OXYGEN  REQUIREMENTS. — Is  a  facultative  anaerobe.  No  growth 
under  mica  plates,  but  can  grow  in  the  body.  The  presence  of  air  is 
necessary  to  the  production  of  the  pigment,  pyocyanin  (p.  116). 

TEMPERATURE. — Grows  at  ordinary  temperature;  best  at  37°. 

BEHAVIOR  TO  GKLATIN.— Liquefies  rapidly.  Under  anaerobic  con- 
ditions this  property  may  be  lost. 

AEROGENESIS. — Produces  a  characteristic  aromatic  odor.  Hydro- 
gen sulphide,  mercaptan,  butyric  acid,  hydrogen,  carbonic  acid,  etc. 

ATTENUATION. — Artificial  cultures  diminish  in  virulence.  More- 
over, variation  in  pigment  production  will  be  met  with.  Even,  color- 
less varieties  have  been  obtained. 

IMMUNITY. — Injection  of  small  amounts  of  the  culture,  or  of  steri- 
lized cultures  induces  immunity. 

PATHOGENESIS. — Subcutaneous  injection,  in  guinea-pigs  and  rab- 
bits, of  about  1  c.c.  of  a  fresh  bouillon  culture  produces  a  rapidly 
spreading  edema,  purulent  inflammation  and  death.  Incase  of  recov- 
ery a  local  abscess  forms.  Intraperitoneal  injections  produce  puru- 
lent peritonitis  and  death.  Bacilli  are  numerous  in  the  tissues,  blood, 
organs,  etc.  Small  amounts  produce  less  marked  results  and  recovery. 
Virulent  cultures  will  kill  rabbits  in  about  2  days  in  a  dose  of  Tfoj-  c.c. 
or  less,  subcutaneously.  Cure  of  animals  infected  with  anthrax  by 
inoculation  with  B.  pyocyaneus.  This  bacillus  was  formerly  consid- 
ered as  a  harmless  form,  accidentally  introduced  into  wounds.  While, 
in  general,  it  is  not  markedly  pathogenic  for  man,  yet  there  are  times 
when  it  may  take  on  toxic  properties. 

DIAGNOSIS.— It  is  to  be  distinguished  from  the  ordinary  fluorescing 
bacteria.  When  an  agar  culture  of  B.  pyocyaneus  is  whipped  up 
with  chloroform,  the  latter  becomes  blue.  Isolated  colonies  in  deep 
gelatin  liquefy,  whereas  those  of  the  fluorescing  bacilli  do  not. 

362 


DRAWINGS. 


sea 


Streptococcus  Pyogenes,  Rosenbach  (1884). 
STREPTOCOCCUS  ERYSiPELATis,  Fehleisen  (J.883). 

ORIGIN. — In  abscesses,  pyemia,  puerperal  fever,  erysipelas  and 
many  other  diseases  as  indicated  below.  In  erysipelas  it  is  found 
in  the  lymphatic  vessels  of  the  diseased  skin,  and  only  very  rarely  in 
the  blood  and  internal  organs.  Streptococci  are  found  in  the  mouth 
and  sputum;  on  the  mucous  membranes  of  the  nose,  urethra,  vagina, 
etc.;  frequently  present  in  mixed  or  secondary  infections. 

FORM. — Small,  spherical  cells  which  may  grow  in  pairs  or  in 
short  chains  of  6-8;  not  infrequently,  as  when  grown  in  bouillon,  the 
•chains  may  consist  of  a  hundred  or  more  cells. 

MOTILITY. — Has  no  motion. 

SPORULATION.— None. 

ANILIN  DYES.— Stain  readily;  Gram's  method  is  applicable. 

GROWTH. — Is  readily  obtained  on  various  media,  even  at  ordinary 
temperature,  but  the  growth  is  slow  and  limited.  Alkaline  reaction 
is  necessary.  Glycerin  agar  is  very  useful.  1  part  of  ordinary 
bouillon  and  2  parts  of  human  blood-serum  yields  the  best  medium. 
In  this  the  virulence  is  unaltered.  2  parts  of  bouillon  and  1  part  of 
ascitic  fluid  is  useful  though  not  as  good  as  the  preceding. 

Plates. — On  gelatin  the  colonies  develop  rather  slowly,  forming  minute  oval  or  round, 
yellowish-brown,  finely  granular  colonies,  which  are  sharply  bordered  and  usually  show 
concentric  rings.  Strface  colonies  remain  small.  No  liquefaction.  On  agar  plates,  devel- 
oped in  the  incubator,  it  forms  delicate,  grayish,  translucent,  drop-like  colonies. 

Stab  culture. — In  gelatin  the  growth  is  quite  characteristic.  Along  the  line  of  inocu- 
lation a  row  of  minute  colonies  forms,  which  usually  remain  separate,  but  may  fuse 
together,  giving  rise  to  a  continuous  line.  Scarcely  any  growth  forms  on  the  surface. 

Streak  culture.— On  agar  or  blood-serum,  it  develops  as  minute,  scarcely  visible, 
round  colonies  which  resemble  minute  dew-drops.  They  do  not  tend  to  spread. 

Bouillon.— At  37°  becomes  diffusely  clouded  and  a  slight,  whitish  sediment  forms.  As 
a  rule,  however,  the  liquid  does  not  cloud  but  remains  clear,  in  which  case  the  growth 
•occurs  on  the  walls  and  bottom  of  the  tube. 

Milk  is  coagulated.    Acid  is  produced.    Growth  on  potato  is  doubtful. 

OXYGEN  REQUIREMENTS.  —Is  a  facultative  anaerobe. 

TEMPERATURE.— Grows  slowly  at  room  temperature;  best  at  30-37°. 

BEHAVIOR  TO  GELATIN. — Does  not  liquefy. 

ATTENUATION. — The  virulence  of  the  organism  is  subject  to  con- 
siderable variation,  even  when  taken  directly  from  a  case  of  the 
•disease.  Artificial  cultures  soon  become  attenuated  and  will  die  out 
unless  transplanted  every  few  weeks.  The  virulence  and  vitality  can 
be  preserved  best  by  sealing  the  heart-blood  of  an  infected  rabbit  in 
tube  pipettes  (see  p.  279).  Vitality  is  also  well  preserved  in  gelatin 
stab  culture;  or  on  agar  to  which  human  blood  has  been  added.  By 
successive  passage  through  animals,  or  by  the  sac  method,  the  viru- 
lence can  be  increased  so  that  virtually  a  single  cell  is  fatal  to  a  rab- 
bit. Thus,  cultures  have  been  obtained  of  which  one  hundred  mil- 
lionth of  a  c.c.  was  invariably  fatal;  whereas,  a  one  hundred  thousand 
millionth  would  kill  1  or  2  out  of  4  rabbits.  The  virulence  can  be 
also  increased  by  injection  together  with  Proteus  vulgaris. 

IMMUNITY,  PATHOGENESIS. — See  p.  383. 

INFECTION. — Undoubtedly  through  wounds  or  injuries  of  the  skin. 
^  DIAGNOSIS.— The  detection  of  streptococci  in  blood  can  be  effected 
by  simple  or  by  Gram's  stain,  supplemented  by  culture  on  glycerin  agar. 
It  is  better  to  add  2  c.c.  of  the  fresh  blood  to  4  c.c.  of  agar  and  to 
make  Petri  plates  with  this  mixture  (37°).  Intra-peritoneal  injection 
into  white  mice;  intravenous  into  rabbits. 

364 


DRAWINGS.  365- 


Staphylococcus  Pyogenes  Aureus,  Rosenbach  (1884). 

GOLDEN  PUS  PRODUCING  COCCUS. 

ORIGIN. — One  of  the  most  common  organisms  in  pus-^tn  about  80 
per  cent.  Found  on  the  skin,  in  saliva,  air,  water,  dust  and  soil. 

FORM. — Small  cocci,  arranged  in  irregular  groups  (Pig.  10  e,  p.  44); 
may  grow  single  or  form  diplococci.  Size  varies  with  the  medium. 

MoTiLiTY.--Has  no  motion. 

SPORULATION. — No  spores  observed.  Possesses  a  high  degree  of 
resistance  to  desiccation,  heat,  chemicals,  etc. 

ANILIN  DYES. — Stain  readily;  so  does  Gram's  method. 

GROWTH. — Is  rapid. 

Plates. — On  gelatin  plates  the  colonies  are  round,  with  sharp  smooth  borders,  very 

¥-anular  and  of  a  dark-brown  or  yellow  color.  The  gelatin  is  liquefied  somewhat  rapidly, 
he  yellowish  colony  lies  in  the  center  of  a  broad  liquefied  disc.  On  agar  the  surface  colon- 
ies are  bright  yellow  in  color. 

Stab  culture. — In  gelatin  development  takes  place  along  the  entire  line  of  inoculation, 
forming  a  finger-shaped  liquefaction.  The  growth  settles  to  the  bottom  as  a  yellowish 
deposit  while  the  liquid  above  remains  clouded  for  some  time.  Peculiar  acid  odor. 

Streak  culture. — On  agar  it  forms  a  moist,  glistening  orange-yellow  covering.  On 
potato  the  growth  is  excellent,  forming  a  thick,  moist  yellow  mass.  Peculiar  odor  present. 

Bouillon. — A  slight  cloud  permeates  the  liquid  and  eventually  a  yellow  sediment 
forms.  Lactic  and  other  acids  develop. 

Milk. — Coagulation  results  and  the  casein  is  then  slowly  peptonized. 

OXYGEN  REQUIREMENTS. — Is  a  facultative  anaerobe.  Pigment  for- 
mation depends  on  presence  of  oxygen. 

TEMPERATURE. — Grows  at  ordinary  temperature:  best  at  30-37°. 

BEHAVIOR  TO  GELATIN. — Liquefies  rapidly. 

ATTENUATION. — The  virulence  is  rapidly  decreased  on  artificial 
media.  The  vitality  is  not  decreased,  even  in  very  old  cultures. 

IMMUNITY. — Repeated  injections  of  small  doses  of  dead  or  living 
cultures  immunize.  The  serum  of  such  animals  seems  to  protect. 

PATHOGENESIS. — Pure  cultures  applied  to  the  unbroken  skin  in 
man  produced  suppuration  'and  carbuncles  (p.  261).  Subcutaneous  ap- 
plication in  mice,  rabbits,  and  guinea-pigs  induces  local  abscesses. 
Intraperitoneal  and  intravenous  injections  produce  fatal  results  with 
formation  of  minute  abscesses  in  the  different  organs  and  tissues — 
pyemia.  Purulent  peritonitis  may  result  and  the  cocci  are  present 
in  the  leucocytes  in  enormous  numbers.  The  organism  is  present  in 
the  blood  as  well  as  in  the  internal  organs.  Intravenous  injection  of 
potato  cultures  induces  ulcerative  endocarditis.  Osteomyelitis  re- 
sults when  the  bones  of  the  leg  are  first  fractured.  It  is  especially 
pathogenic  for  man.  The  virulence  of  the  culture  and  the  avenue  of 
infection  are  of  great  importance.  The  cells  contain  the  active 
toxin  which  is  not  destroyed  by  heat.  This  is  seen  in  the  fact  that 
sterilized  cultures  induce  suppuration. 

The  golden  sta.phylococcus  is  by  far  the  most  common  cause  of 
pus  formation.  It  may  occur  alone,  or  may  be  associated  with  the 
white  or  lemon  staphylococcus,  or  with  streptococci  and  other  bac- 
teria. It  is,  therefore,  to  be  expected,  as  a  rule,  in  acute  abscesses 
and  boils;  also  in  angina,  empyema,  otitis  and  osteomyelitis  Its 
presence  in  endocarditis  and  pyemia  has  been  referred  to. 

INFECTION.— Usually  through  scratches  and  wounds.  May  pene- 
trate the  uninjured  skin. 

DIAGNOSIS. — A  microscopical  and  cultural  examination  of  the  pus 
will  reveal  the  characteristic  organism  if  present.  In  suspected 
pyemia  2  c.c.  of  the  blood  may  be  plated  (p.  364). 

In  suppuration  other  staphylococci  may  be  found,  as  the  8.  pyo- 
genes albus  and  the  8.  pyogenes  citreus.  These  perhaps  are  less  fre- 
quent and  less  virulent.  The  cultural  properties  are  practically  the 
same  with  the  exception  of  the  difference  in  pigment. 

366 


DRAWINGS.  367 


Micrococcus  Gonorrhceae,  Neisser  (1879). 

GONOCOCCUS,    DIPLOCOCCUS   OF  GONORRHEA. 

ORIGIN. — Constant  in  gonorrheal  discharges.  The  disease  may- 
affect  any  mucous  membrane:  urethra,  bladder,  rectum,  conjunctiva, 
uterus,  etc.  The  organism  is  present  in  ophthalmia  neonatorum,. 
gonorrheal  ophthalmia,  gonorrheal  rheumatism  and  salpingitis.  It 
may  even  cause  endocarditis  and  general  septicemia. 

FORM. — "Biscuit-shaped"  micrococci  which  are  usually  in  pairs,, 
with  the  flattened  surfaces  facing-  each  other  (Fig.  10  a,  p.  44).  The 
diplococcus  is  usually  grouped  in  masses  of  20-40  or  more  cells.  The 
pus  cells  are  frequently  invaded  and  filled  by  the  gonococci. 

In  pure  culture  the  typical  diplococcus  form  is  rarely  present. 
Rounded  cubes  in  irregular  masses  are  frequent. 

MOTILITY. — Has  no  real  motion. 
SPORULATION. — Not  known. 

ANILIN  DYES. — Stain  readily.  It  is  decolored  by  Gram's  method, 
especially  if  the  specimen  is  not  washed  in  water.  Excellent  double 
stains  can  be  obtained  if  the  specimen  is  first  treated  with  dilute 
eosin  or  safranin  and  then  with  Loffler's  methylene  blue,  or  with 
methyl  violet.  Mathylene  blue  is  best  adapted  for  staining  cover- 
glasses  of  gonorrheal  pus. 

GROWTH. — Behaves  as  a  strict  parasite,  and  hence  can  be  grown 
only  under  very  special  conditions  of  soil  and  temperature.  Requires 
an  albuminous  medium  (see  p.  384). 

Plates. — No  growth  on  gelatin  or  agar  plates.  On  cigar-serum  plates  (p.  384)  the 
colonies  appear  in  2^  hours  as  transparent,  finely  granular  points,  which  show  an  indented 
border.  They  may  become  yellowish  with  coarsely  granular  center. 

Stab  culture.— In  agar-serum  a  thin  delicate  growth  forms  along  the  line  of  inocula- 
tion. A  similar  film  spreads  over  the  surface.  Ascites-agar  (1:2)  is  excellent. 

Streak  culture. — On  inclined  agar-serum  it  yields  a  glistening,  grayish  white  growth. 
On  blood-agar  it  forms  small  transparent  discrete  colonies  which  resemble  those  of  the 
influenza  bacillus.  On  rabbit  serum  the  colonies  are  minute,  transparent,  roundish  with  a 
raised  center;  slimy  character. 

Ascitic  fluid  and  ordinary  bouillon  (i  :  3)  gives  at  36°  an  excellent  growth.  At  first  it 
becomes  cloudy  and  a  fine  deposit  forms;  eventually,  a  slight  creamy  or  visc9us  film 
fcnrnis  on  the  surface;  long  filaments  descend  into  the  liquid.  The  culture  dies  out  in  about 
a  week. 

OXYGEN  REQUIREMENTS. — It  is  an  aerobic  organism. 

TEMPERATURE.— Growth  at  32°  to  38°.  A  temperature  of  35°-37° 
should  be  maintained,  otherwise  it  will  soon  die  out.  Above  40°  it  is 
easily  killed.  Likewise  in  a  few  hours  at  15°. 

ATTENUATION. — Dies  out  very  rapidly  on  some  media.  May  live 
on  rabbit  serum  at  36°  for  3-4-8  weeks. 

PATHOGENESIS. — Pure  cultures  of  the  gonococcus  produce  typical 
gonorrhea  when  introduced  into  the  healthy  urethra.  The  toxin  has 
a  like  effect.  Gonorrheal  pus  may  be  inoculated  on  the  mucous  mem- 
branes of  animals  without  the  least  effect.  Intraperitoneal  injection 
into  mice  produces  a  non-fatal  purulent  peritonitis.  The  gonococcus 
is,  therefore,  pathogenic  only  for  man. 

DIAGNOSIS. — See  p.  384. 

368 


DRAWINGS.  369 


24 


Micrococcus  Tetragenus,  Gaffky  (1881). 


ORIGIN. — First  obtained  from  the  contents  of  a  tubercular  lung- 
Cavity;  present  in  normal  saliva  (26  times  out  of  111  cases,  Miller), 
rather  common  in  sputum  of  tubercular  persons.  Has  been  found  in 
a  few  instances,  as  the  only  organism  present  in  acute  abscesses.  A 
similar  non-pathogenic  organism  which,  however,  cannot  be  grown 
^artificially,  may  be  present  in  the  mouth. 

FORM.  —Large  cocci,  which  in  pure  cultures  on  artificial  media 
are  either  single  or  in  pairs,  or  in  irregular  groups.  In  the  animal  body 
it  forms  perfect  tetrads,  which  are  surrounded  by  a  wide  colorless 
capsule  (Fig.  5  &,  p.  29). 

MOTILTTY. — None. 

SPORULATION.  — None. 

ANILIN  DYES. — Stain  readily.     Gram's  method  is  applicable. 

GROWTH. — Is  rather  slow. 

Plates.— The  colonies  which  develop  on  the  gelatin  plate  are  round  or  oval,  slightly 
granular,  yellowish  and  sharp  bordered.  No  liquefaction.  The  surface  colonies  are  white, 
elevated  and  thick. 

Stab  culture. — Along  the  line  of  inoculation,  in  the  gelatin  tube,  the  growth  develops 
either  as  a  row  of  white  dots  or  as  a  continuous  white  line.  On  the  surface  a  characteristic 
moist,  white,  thick  mass  forms. 

Streak  culture.— On  agar  it  usually  develops  as  discrete  sharply  defined,  round,  white 
colonies.  At  times  the  growth  may  be  confluent.  On  potato  it  forms  a  thick,  slimy  cover- 
ing, which  can  be  drawn  out  into  long  threads. 

OXYGEN  REQUIREMENTS. — It  is  a  facultative  anaerobe. 

TEMPERATURE. — Grows  well  at  ordinary  temperature;  better  in 
the  incubator 

BEHAVIOR  TO  GELATIN.— Does  not  liquefy. 

ATTENUATION. — Cultures  grown  for  years  on  artificial  media  even- 
tually become  attenuated. 

PATHOGENESIS. — White  mice  and  guinea-pigs  are  susceptible. 
House  and  field  mice  are  usually  insusceptible,  while  rabbits  and  dogs 
are  immune.  A  subcutaneous  application  or  intraperitoneal  injec- 
tion kills  white  mice  in  from  3-10  days.  The  blood-vessels  of  the  kid- 
ney, spleen,  liver,  lungs,  etc.,  are  full  of  the  tetrads  which  are  in- 
vested by  capsules.  Subcutaneous  injection  into  guinea-pigs  pro- 
duces usually  a  local  abscess;  whereas  an  intraperitoneal  injection  is 
followed  by  a  purulent  peritonitis.  The  exudate  in  this  case  is  rich 
in  capsulated  forms.  The  frequent  presence  of  this  organism  in 
tuberculous  cavities  indicates  that  it  plays,  as  well  as  streptococci 
and  other  bacteria,  an  active  part  in  the  destruction  of  the  lung 

tissue. 

370 


DRAWINGS.  371 


Spirillum  Obermeieri,  Obermeier  (1873). 

SPIRILLUM  OF  RELAPSING    OR    RECURRENT  FEVER; 
SPIROCHAETE  OBERMEIERI. 

ORIGIN. — It  is  always  and  exclusively  present  in  the' 
blood  of  relapsing"  fever  patients,  and,  is  especially  found 
during  the  febrile  paroxysms  at  which  time  it  may  be 
present  in  large  numbers.  A  similar,  closely  related  spiril- 
lum is  found  in  the  Trans-Caucasus  in  the  blood  of  geese 
affected  with  a  disease  which  resembles  somewhat  the 
recurrent  fever  of  man  (S.  anserini,  SakharofT). 

FORM. — Delicate,  flexible,  long,  wavy  spirals.  May 
have  10-20  windings.  Length  is  usually  from  16  to  30  />.. 
They  are  much  narrower  than  the  comma  bacillus. 

MOTILITY. — Actively  motile.  Flagella  have  not  been 
demonstrated.  It  preserves  its  motility  at  the  ordinary 
temperature  for  many  days. 

SPORULATION. — This  has  not  been  observed. 

ANILIN  DYES. — It  is  stained  rapidly  and  intensely  by  the 
ordinary  dyes  (p.  281).  Gentian  violet  is  useful  for  this 
purpose.  Gram's  method  is  not  applicable. 

GROWTH. — It  is  an  obligative  parasite.  It  has  not  been 
successfully  cultivated  outside  of  the  living  body.  They 
may  live  in  the  blood  of  a  leech  for  some  days. 

IMMUNITY. — One  attack  does  not  protect  against  another. 

PATHOGENESIS. — Inoculation  of  healthy  individuals  with 
blood  which  contains  these  spirals  produces  typical  relap- 
sing fever,  which  is  accompanied  by  the  presence  of  the 
characteristic  spirals.  The  disease  can  also  be  transmitted 
to  monkeys,  but  the  animal  usually  recovers.  In  monkeys 
from  which  the  spleen  has  been  removed  the  spirilla  de- 
velop in  enormous  numbers  in  the  blood,  and  death  results1. 
Although  it  has  not  been  isolated  and  grown  in  pure  cul- 
ture, yet  the  constant  presence  of  the  organism  in  this  dis- 
ease leads  to  the  accepted  belief  that  it  is  the  cause.  This 
is  also  true  of  the  leprosy  bacillus,  the  plasmodium  of  ma- 
laria and  the  ameba  coli  of  dysentery. 

DIAGNOSIS. — The  spirillum  is  easily  detected  if  present 
in  large  numbers.  Otherwise  it  will  require  a  very  careful 
examination.  The  blood  should  be  examined  during  the 
attack. 

1  Consult  Soudakewitch,  Annales  de  1'Institut  Pasteur,  5,  1891, 
p.  545.  Plates  XIV-XVII. 

372 


DRAWINGS.  373 


Bacillus  Cholerae  Gallinarum,   Perroncito,  Pasteur  (1880). 

CHICKEN    OR    FOWL    CHOLERA;   CHOLERA  DES  POULES   (Fr.)', 
HUHNER-,GEPLUGEL-CHOLERA   (Germ.}. 

ORIGIN.— In  the  blood,  organs  and  excreta  of  chickens  which  have 
the  disease.  A  somewhat  similar  disease  of  chickens,  occurring  in 
Russia,  has  been  shown  to  be  due  to  the  Vibrio  Metchnikovi  which 
resembles  very  closely  the  vibrio  of  Asiatic  cholera.  A  similar,  if 
not  identical  organism  has  been  isolated  by  Koch  from  water  and  de- 
signated as  the  B.  of  rabbit  septicemia. 

FORM. — Small  short  rods  which  have  rounded  ends  and  are  fre: 
quently  in  pairs,  rarely  in  long  threads.  At  times  the  form  is  almost 
that  of  a  coccus. 

MOTILITY. — It  has  no  motion. 

SPORULATION. — Spores  have  not  been  observed.  Nevertheless,  it 
possesses  considerable  power  of  resistance  and  can  withstand  the 
acidity  of  the  gastric  juice. 

ANII.IN  DYES. — Usually  stain  the  ends  first  while  the  center  re- 
mains uncolored — bi-polar  stain.  The'  appearance  then  is  that  of  a 
diplococcus.  On  more  intense  staining  the  entire  rod  becomes  col- 
ored. Gram's  method  is  not  applicable. 

GROWTH.     Is  rather  slow. 

Plates. — Colonies  appear  in  a  few  days  on  geldtin  plates  as  minute  white  dots  which, 
under  the  microscope,  are  seen  to  be  roundish  plates  with  sharp,  smooth  borders.  The 
contents  are  finely  granular,  show  concentric  rings  and  are  yellowish  in  color.  No  lique- 
faction. 

Stab  culture. — Forms  \ngelatin,  a  delicate  white  line  or  row  of  dots  along  the  line  of 
inoculation.  On  the  surface  it  forms  a  delicate  whitish  growth  which  spreads  very  slowly. 

Streak  culture. — On  agar  it  develops  as  a  thick,  glistening,  grayish-white  mass.  No 
growth  on  potato  at  ordinary  temperature,  but  in  the  incubator  in  a'few  days  it  gives  rise 
to  a  yellowish-gray  transparent  wax-like  covering. 

Bouillon. — Diffuse  cloudiness  and  a  slight  deposit.    Indol  is  formed. 

Milk. — Is  slowly  coagulated — acid  production. 

OXYGEN  REQUIREMENTS. — Is  a  facultative  anaerobe. 

TEMPERATURE. — Grows  at  ordinary  temperature  and  also  in  the 
incubator. 

BEHAVIOR  TO  GELATIN. — Does  not  liquefy. 

ATTENUATION.— Artificial  cultures  soon  lose  their  virulence.  The 
virulence  can  be  increased  remarkably  by  successive  passage  through 
animals,  and  by  the  collodium  sac  method.  In  sealed  tubes  (Figs.  61, 
62)  the  virulence  is  preserved. 

IMMUNITY. — Is  produced  in  chickens  and  pigeons  by  inoculation 
with  first  and  second  vaccines.  It  was  in  connection  with  this  dis- 
ease that  vaccination  by  means  of  attenuated  cultures  was  discovered 
(Pasteur,  1880). 

PATHOGENESIS. — Chickens,  geese,  pigeons,  sparrows,  mice  and  rab- 
bits are  susceptible  to  subcutaneous  inoculation.  Guinea-pigs,  sheep, 
horses  are  less  susceptible  and  only  local  abscesses  form.  Dogs  and 
cats  are  immune.  After  death  the  bacilli  are  found  distributed 
throughout  the  body — a  true  septicemia. 

INFECTION. — Usually  results  in  chicken  through  the  food.  It  may 
possibly  occur  through  scratches  and  wounds. 

DIAGNOSIS. — Stained  preparations  of  the  blood  (p.  281)  will  show 
the  typical  short  rods  in  enormous  numbers.  Absence  of  motion,  the 
behavior  to  milk,  etc.,  and  the  animal  experiment  will  distinguish  it 
from  similar  organisms. 

374 


DRAWINGS.  375 


Bacillus  Cholerse  Suis. 
HOG  CHOLERA;    SWINE-PLAGUE  of  Billings;  SCHWEINEPEST  (G?erm.); 

CHOLERA  DU  PORC,   PNEUMO-ENTERITE  (Fr.). 

ORIGIN. — In  the  blood,  organs  and  intestinal  contents  of  swine 
that  die  of  hog  cholera. 

FORM. — Short,  small  rods,  like  those  of  chicken  cholera.  On  some 
media,  as  gelatin,  it  may  form  long  rods.  Occurs  single  or  in  pairs. 

MOTILITY. — It  is  actively  motile  and  has  several  long,  wavy  flag- 
ella.  Shows  no  motion  in  serum  or  in  blood. 

SPORULATION. — Not  observed. 

ANILIN  DYES. — At  first  impart  a  bi-polar  stain,  but  on  sufficient 
exposure  the  entire  -rod  is  colored.  Not  stained  by  Gram's  method. 

GROWTH.— Is  fairly  rapid. 

Plates. — In  a  couple  of  days  colonies  develop  on  gelatin  plates.  The  deep  colonies 
are  very  small,  yellowish-brown  and  spherical.  The  surface  colonies  spread  slightly.  No 
liquefaction. 

Stab  culture.— Shows  along  the  line  of  inoculation  a  white  line  or  row  of  colonies, 
while  on  the  surface  of  the  gelatin  a  thin,  very  slowly  spreading  growth  forms. 

Streak  culture.— On  agar  forms  a  moist  grayish-white  growth,  without  any  special 
characteristics.  On  potato  a  straw-yellow  growth  develops,  resembling  somewhat  that  of 
glanders. 

Bouillon.— Diffuse  cloudiness  of  the  liquid;  a  partial  film  forms  on  the  surface.  Indol 
and  phenol  are  not  formed.  Milk. — Is  not  coagulated. 

OXYGEN  REQUIREMENTS. — It  is  a  facultative  anaerobe. 
,  TEMPERATURE. — Grows  well  at  ordinary  temperature.    Best  at  37°. 

BEHAVIOR  TO  GELATIN. — Does  not  liquefy. 

AEROGENESIS. — Glucose  is  fermented. 

ATTENUATION. — Artificial  cultures  retain  their  virulence  quite 
well.  The  virulence  can  be  readily  increased  Jby  repeated  passage 
through  rabbits.  It  is  then  very  fatal  to  pigeons. 

IMMUNITY. — Can  be  produced  experimentally  by  inoculation  with 
filtered  cultures;  with  repeated  small  doses  of  blood,  previously 
heated  to  54-58°,  from  infected  rabbits  (?)>  Blood  serum  of  immun- 
ized animals  protects. 

PATHOGENESIS.— Hogs,  mice,  rabbits  and  guinea-pigs  are  highly 
susceptible;  pigeons  are  less  susceptible,  while  chickens,  sheep  and 
calves  are  immune.  %  c.c.  of  bouillon  culture  injected  subcutane- 
ously  into  rabbits  kills  in  about  four  days.  Bacilli  distributed  every- 
where. White  necrotic  areas  in  the  liver.  Hemorrhagic  infiltrations 
are  common. 

INFECTION.— Results  through  the  food.  The  'hog  is  the  only  ani- 
mal that  naturally  contracts  the  disease. 

DIAGNOSIS. — As  a  rule  the  bacillus  can  be  isolated  from  the  heart- 
blood -and  organs.  In  chronic  cases,  because  of  secondary  infection, 
diverse  bacteria  may  be  present.  .  Isolation  may  be  facilitated  by 

inoculating  a  rabbit  with  the  suspected  material. 

376 


DRAWINGS.  377 


Bacillus  Rhusiopathiae  Suis,  Pasteur  (1882). 
HOG  ERYSIPELAS;  ROUGET  (JPV.);  SCHWEINEROTHLAUF  (Germ.). 

ORIGIN. — In  the  blood,  internal  organs,  etc.,  of  swine  infected 
with  the  disease. 

FORM. — In  the  body  it  occurs  as  very  small,  narrow  rods  resem- 
bling- needle-shaped  crystals.  On  some  media,  as  glycerin  agar,  the 
slender  rods  may  be  quite  long,  and  may  show  a  slight  bend.  Are 
usually  single,  but  may  occur  in  pairs  and  even  in  threads. 

MOTILITY.— It  has  no  motion. 

SPORULATION. — Spore  formation  is  not  known. 

ANILIN  DYES.— Stain  readily.      Gram's  method,  excellent  results. 

GROWTH.  -Is  rather  slow  but  extremely  characteristic. 

Plates.— On  gelatin  plates  the  colonies  are  very  characteristic  and  appear  as  diffuse 
cloudy  patches  which  are  sometimes  difficult  to  see.  Little  or  no  surface  growth.  No 
liquefaction. 

Stab  culture. -In  gelatin  is  likewise  very  characteristic.  The  growth  develops 
along  the  line  of  inoculation  as  a  delicate,  cloud-like  radiating  column.  As  the  culture  be- 
comes old  a  depression  forms  at  the  top,  due  to  slow  liquefaction  and  corresponding  evap- 
oration. Sometimes  liquefaction  can  be  observed.  The  vitality  is  maintained  longest  in 
gelatin  cultures. 

Streak  culture. — On  agar  and  on  blood-serum  it  forms  a  scarcely  visible  thin  film  or 
group  of  colonies.  Glycerin  agar  is  best  and  on  this  the  vitality  is  prolonged.  No  growth 
on  potato  except  under  anaerobic  conditions. 

Bouillon. — A  very  delicate  diffuse  cloudiness  forms  which  can  best  be  seen  on  slight 
agitation.  Resembles  the  bouillon  culture  of  the  tetanus  bacillus.  No  indol  is  formed. 

OXYGEN  RKQUIREMKNTS. — Is  a  facultative  aerobe.  Best  growth 
under  anaerobic  conditions,  which,  moreover,  preserve  the  virulence. 

TEMPERATURE. — Grows  slowly  at  ordinary  temperature.  Best  at  36° . 

BEHAVIOR  TO  GELATIN. — Does  not  perceptibly  liquefy  gelatin. 

AEROGENESIS. — Produces  hydrogen  sulphide  in  pure  cultures,  and 
in  the  body.  This  gas  is  also  produced  by  the  anaerobic  bacteria  and 
to  a  less  extent  by  nearly  all  pathogenic  bacteria.  .  ,  ' 

ATTENUATION.— Old  cultures  become  attenuated;  this  result  can 
also  be  obtained  by  growing  the  virulent  germ  at  high  temperatures, 
about  42°,  for  some  time  (Pasteur).  Passage  through  rabbits  attenu- 
ates; whereas  passage  through  pigeons  increases  the  virulence  with 
reference  to  swine. 

IMMUNITY.— By  inoculation  with  attenuated  culture— first  and 
second  vaccine  of  Pasteur — perfect  immunity  can  be  produced.  One 
attack  of  the  disease  confers  immunity.  The  serum  of  immunized 
animals  is  anti-infectious. 

PATHOGENESIS.— Swine,  rabbits,  pigeons,  white  mice,  rats  and 
house  mice  are  susceptible.  Death  in  3  to  5  days.  Field  mice,  guinea- 
pigs,  dogs,  cats,  chickens  and  ducks  are  insusceptible.  Bacilli  distri- 
buted throughout  the  organism  (septicemia).  They  are  very  numerous 
in  the  blood:  single  or  in  pairs;  very  often  can  be  seen  to  be  enclosed 
in  leucocytes. 

INFECTION.— Occurs  naturally  in  swine  through  the  food.  The 
bacillus  is  always  present  in  the  feces. 

DIAGNOSIS.— The  bacillus  can  be  readily  detected  in  the  blood  and 
especially  in  the  spleen  by  Gram's  method.  The  cultural  character- 
istics will  distinguish  it  from  all  other  organisms  except  that  of 
mouse  septicemia.  Inoculation  of  mouse  or  rabbit. 

378 


DRAWINGS.  379 


Bacillus  Murisepticus,  Koch  (1878). 
MOUSE  SEPTICEMIA;  MAUSESEPTIKAMIE  (Germ.). 

ORIGIN.— From  mice  after  inoculation  with  putrid  blood.  It  is 
widely  distributed  in  water,  soil,  etc. 

FORM. — The  rods  are  narrower  and  thinner  than  those  of  the 
rouget  bacillus,  but  otherwise  resemble  the  latter  very  much. 

MOTILITY. — Appears  to  possess  motion,  but  is  really  non-motile. 

SPORULATION. — Round,  glistening-  bodies  form  within  the  cells  as 
in  the  case  of  rouget.  They  have  nothing-  to  do  with  spores. 

ANILIN  DYES. — Stain  rapidly.    Gram's  method  is  applicable. 

GROWTH.— Is  rather  slow  and  resembles  very  closely  that  of  the 
roug-et  bacillus. 

Plates.— The  colonies  on  the  gelatin  plate  resemble  those  of  the  rouget  bacillus,  ex- 
•cept  that  they  spread  [somewhat  more  rapidly  and  are  more  delicate  and  transparent  in 
appearance. 

Stab  culture.— Shows  this  distinction  in  growth  quite  sharply.  While  the  cloudy 
growth  of  the  rouget  bacillus  is  dense  and  somewhat  limited  to  the  line  of  inoculation,  that 
of  the  mouse  septicemia  bacillus  is  lighter  and  spreads  readily  throughout  the  entire  gela- 
tin. This  difference  is  clearly  seen  in  young  cultures. 

Streak  culture.— On  agar  the  growth  is  scarcely  to  be  distinguished  from  that  of  the 
rouget  bacillus.  Glycerin  agar  is  most  suitable. 

Bouillon. — The  bacillus  develops  a  growth  similar  to  that  of  the  bacillus  of  rouget. 

OXYGEN  REQUIREMENTS.— It  is  a  facultative  aerobe  and  hence  grows 
best  when  air  is  excluded. 

TEMPERATURE. — It  grows  well  at  the  ordinary  temperature.  Opti- 
mum about  35-37°. 

BEHAVIOR  TO  GELATIN. — Ordinarily  no  liquefaction  can  be  observed. 
Sometimes,  however,  the  gelatin  gradually  softens. 

AEROGENESIS. — Produces  less  hydrogen  sulphide  than  the  rouget 
bacillus. 

ATTENUATION.— Old  cultures  possess  diminished  virulence. 

IMMUNITY.— Rabbits  that  recover  after  one  inoculation  with  the 
pure  culture  are  rendered  immune  against  subsequent  inoculation. 
They  are  at  the  same  time  immune  against  the  rouget  bacillus. 

PATHOGENESIS.— White  mice,  house  mice,  pigeons,  sparrows  and 
rabbits  are  susceptible.  Chickens,  guinea-pigs  and  field  mice  are 
wholly  immune.  After  death  the  bacilli  are  distributed  throughout 
the  body,  single  or  in  pairs,  and  are  frequently  inclosed  in  leucocytes. 

DIAGNOSIS.— In  cultural,  morphological  and  pathogenic  properties 
it  resembles  very  closely  the  preceding  organism.  So  much  so  that 
it  is  commonly  considered  to  be  an  attenuated  form  of  the  rouget 

bacillus. 

380 


DRAWINGS.  381 


382  BACTERIOLOGY. 

Colon  Bacillus  (p.  352). 

DIAGNOSIS. — The  motility  of  the  colon  bacillus  distinguishes  it 
from  the  aerogenes  group  of  bacteria,  which  includes  well  known 
intestinal  bacteria,  such  as  the  B.  lactis  aerogenes,  and  the  B.  coli 
immobilis.  The  latter  forms  tend  to  produce  convex  raised  growths  on 
the  surface  of  gelatin  in  plate  and  stab  cultures,  whereas  the  motile 
colon  bacillus  is  more  likely  to  give  a  thin  spreading  growth.  The 
variable  motility  of  the  colon  bacillus,  the  influence  of  temperature, 
and  the  consistency  of  the  gelatin  influence  the  cultural  characteris- 
tics to  such  an  extent  as  to  make  this  distinction  of  but  little  value. 

The  colon  bacillus  is  to  be  especially  distinguished  from  the 
typhoid  and  similar  bacilli.  The  distinctions  are  given  at  length  in 
Chapter  XIII.  Acid,  gas  and  indol  production  as  well  as  coagulation 
of  milk  are  important  characteristics. 

Eberth  Bacillus  (p.  354). 

INFECTION. — Commonly  takes  place  through  the  mouth  by  means 
of  water,  food,  contact  with  soiled  articles,  etc.  Air  transmission, 
as  fine  dust,  is  possible.  Danger  from  flies  and  other  insects.  In 
later  stages  other  organisms  as  streptococci  frequently  appear — 
mixed  infection. 

DIAGNOSIS. — The  differentiation  of  the  Eberth  from  the  colon 
bacillus  is  difficult  and  necessitates  a  comparison  of  all  known  reac- 
tions (See  Chapters  XIII  and  XIV).  The  recognition  of  the  disease 
is  based  primarily  upon  the  serum  reaction  (Chapter  XV)  and  to  less 
extent,  as  yet,  upon  the  isolation  of  the  bacillus  from  urine  or  feces. 

Bacillus    Icteroides   (p.  356). 

PATHOGENESIS. — It  is  very  pathogenic  for  white  mice,  rabbits, 
guinea-pigs.  Dogs,  monkeys,  goats,  sheep  and  horses  are  also  sus- 
ceptible. In  guinea-pigs  1  c.c.  of  a  24  hour  bouillon  culture,  intro- 
duced subcutaneously  or  into  the  peritoneal  cavity,  produces  death 
in  from  4-7  days.  Virulent  cultures,  obtained  by  frequent  transplan- 
tations, will  kill  in  less  than  24  hours.  In  the  latter  case  the  bacilli 
are  extremely  numerous  on  the  serous  surfaces,  in  the  blood  and  in- 
ternal organs — septicemia.  When  death  is  delayed  they  are  very 
scarce,  and  little  or  no  peritoneal  effusion  exists. 


STREPTOCOCCI.  383 

DIAGNOSIS.  The  organism  may  be  found,  at  times,  in  the  blood  of 
yellow  fever  patients.  2  c.c.  of  blood  should  be  drawn  (Chapter  XIV), 
added  to  liquefied  agar  or  gelatin  and  plated.  It  may  also  be  streaked 
over  the  surface  of  inclined  agar.  Suspected  colonies  should  be  re- 
plated,  under  conditions  of  temperature,  already  emphasized,  in" 
order  to  obtain  typical  growths  on  g-elatin  and  on  ag-ar.  In  the  ab- 
sence of  characteristic  growth  the  other  cultural  and  morphological 
properties  should  be  tested. 

Black   Plague  (p.  358). 

PATHOGENESIS.— It  is  fatal  to  white  mice  or  rats  in  1-3  days:  to 
guinea-pig's  in  2-5  days;  to  rabbits  in  2-7  days;  to  monkeys  in  2|-5 
days.  The  horse,  pig-eon,  field  mouse,  hog",  cat  and  frogs  are  some- 
what refractory.  Doves,  chickens,  geese,  dogs  and  cattle  are  said  to 
be  immune.  Abscesses  are  frequently  produced  by  the  bacillus  or  by  its 
toxin.  On  post-mortem  the  animals  show  an  extensive  rose-colored 
edema,  enlarged  lymphatic  vessels  and  glands  and  hemorrhagic  con- 
dition of  the  abdominal  walls.  The  minute  rods  are  numerous  in  the 
blood  and  internal  organs— septicemia.  They  may  be  englobulated  in 
leucocytes.  Accidental  inoculation  with  pure  cultures  has  proven 
fatal  to  man. 

INFECTION.— The  disease  naturally  attacks  mice,  rats,  hogs,  buf- 
faloes, flies  and  man.  Rats  and  flies  are  important  as  a  means  of 
spreading  the  disease.  It  may  be  contracted  through  wounds  -inocu- 
lation form;  or  by  inhalation— p?ieimiomc  form;  or  through  the  food — 
intestinal  form. 

DIAGNOSIS.— The  pus  from  the  bubo  will  show  enormous  numbers 
of  the  short,  oval  rods.  In  the  pneumonic  form  they  can  be  detected 
in  the  sputum.  The  cultural  and  morphological  characteristics  will 
be  necessary  to  complete  the  identification.  The  involution  forms, 
according  to  Hankin,  are  especially  marked  when  the  pure  culture 
of  the  organism  is  planted  on  agar  to  which  about  3  per  cent,  of  salt 
has  been  added.  In  24-48  hours  at  37°  large  spherical  or  pear  shaped 
involutions  can  be  found.  Pest  serum  causes  agglutination. 

Streptococci  (p.  364). 

IMMUNITY.— Can  be  established  in  horses  and  other  animals  by  re- 
peated injection  of  filtered  or  of  virulent  cultures.  The  blood-serum  in 
that  case  is  not  protective  against  all  varieties  of  streptococci.  This 


384  BACTERIOLOGY. 

would  indicate  the  existence  of  diverse  species  although  morphologi- 
cally they  may  be  almost  alike. 

PATHOGENESIS. — Man,  rabbits  and  mice  are  susceptible.  Typical 
erysipelas  results  in  man  from  inoculation  with  pure  cultures,  as  in 
cases  of  inoperable  carcinoma,  etc.  Infection  in  young-  rabbits  fre- 
quently gives  rise  to  local  suppuration,  or  to  severe  general  symptoms 
and  death.  Like  the  Frankel  diplococcus,  it  is  a  widely  distributed 
engenderer  of  inflammatory  processes.  It  may  occur  in  connection 
with  other  diseases  as  diphtheria,  scarlet  fever,  typhoid  fever,  pneu- 
monia, tuberculosis,  etc.  (secondary  infection}.  Or,  it  may  cause  a 
primary  infection,  in  which  case  the  results  will  depend  largely  upon 
the  virulence  of  the  germ  and  the  avenue  of  infection.  Thus,  it  is 
the  common  cause  of  puerperal  fever,  erysipelas,  otitis,  endocarditis, 
pericarditis,  pleurisy,  peritonitis  and  pseudo-diphtheria;  of  a  simple 
abscess  or  of  general  sepsis  (pyemia). 

Gonococcus  (p.  368). 

Pure  human  serum  was  used  at  first  f  or  cultivating  this  organ- 
ism. Better  results,  however,  are  obtained  with  human  serum  or 
blood  added  to  agar  or  bouillon  (1:  2).  The  serum  or  blood  may  be 
replaced  by  ascitic  fluid;  1  part  of  latter  fto  2  or  3  parts  of  agar  or 
bouillon.  Coagulated  rabbit  serum  is  said  to  be  as  good  as  human 
blood.  For  its  preparation  see  Chapter  XIV. 

To  isolate  the  gonococcus  the  pus  should  be  stirred  into  liquefied 
agar  (45°)  and  dilutions  in  agar  should  be  made  in  the  usual  way.  The 
tubes  should  each  contain  about  4  c.c.  of  agar.  2c.c.  of  freshly  drawn 
human  blood  (see  Chapter  XIV)  should  then  be  added  to  each  tube, 
and  the  mixture  poured  into  Petri  dishes.  These  should  then  be  kept 
at  35-37  ° .  The  organism  may  also  be  isolated  by  streaking  the  pus  over 
agar  plates  or  inclined  agar  on  which  human  blood  has  been  spread. 
Or,  the  material  may  be  spread  thoroughly  over  inclined  rabbit 
serum.  The  cultures  should  be  placed  at  once  in  the  incubator. 

DIAGNOSIS. — Cover-glasses  should  be  prepared  from  gonorrheal 
pus  while  it  is  fresh,  and  before  it  has  dried  down.  It  is  advisable  to 
dilute  the  drop  of  pus  with  a  drop  or  two  of  water.  The  cover-glasses 
should  not  be  over-heated.  They  should  be  stained  with  simple,  or 
with  Loffler's  methylene  blue.  The  characteristic  form,  grouping, 
presence  in  pus  cells,  and  the  fact  that  it  does  not  stain  by  Gram's 
method  will  serve  to  distinguish  it  from  other  organisms.  It  can  be 
detected  in  blood  by  the  method  given  for  streptococci  (p.  364). 


CHAPTER  XII. 
YEASTS,  MOULDS  AND  STREPTOTRICES. 

The  study  of  micro-organisms  as  the  cause  of  diverse 
fermentations  and  of  disease  would  be  incomplete  if  limited 
to  the  group  of  bacteria.  While  it  is  true  that  the  latter 
are  the  most  frequent  causes  of  such  conditions,  it  must  be 
remembered  that  there  are  other  organisms  entirely  distinct 
from  bacteria  which  can  induce  similar  changes.  The 
yeasts,  moulds  and  the  streptothrix  forms  are  extremely 
important  in  this  respect.  Furthermore,  in  practical  lab- 
oratory work  yeasts  and  moulds  are  frequently  met  with 
as  contaminations  of  plate  or  tube  cultures.  Conse- 
quently, it  is  advisable  to  understand  the  general  charac- 
teristics of  these  organisms  in  order  to  facilitate  their 
recognition. 

Yeasts. 

These  are  microscopic  unicellular  plants  which  differ 
from  bacteria  in  size  and  in  manner  of  multiplication. 
They  are  usually  about  6  P.  thick;  in  other  words,  they  are 
about  as  large  or  even  larger  than  the  red  blood  cell.  Con- 
sequently, a  very  simple  examination  will  be  sufficient  to 
distinguish  the  yeasts  from  the  bacteria. 

While  bacteria  multiply  by  dividing  in  two  equal  parts 
the  yeasts  increase  in  number  by  a  process  of  budding* 
That  is  to  say,  at  some  point  on  the  surface  of  the  cell  a 
minute  protuberance  develops  which  gradually  enlarges  and 
forms  a  daughter  cell.  This  budding  may  occur  at  several 
places  in  the  same  cell  and  the  young  cells,  thus  formed, 

25 


386  BACTERIOLOGY. 

may  remain  attached  to  the  original  one.  It  is  because  of 
this  peculiarity  that  yeasts  are  known  as  budding-  fungi  or 
blastomycetes. 

The  yeast-cells  are  large,  oval  or  roundish  bodies,  but 
under  special  conditions,  as  when  grown  deep  in  gelatin, 
they  may  elongate  and  give  rise  to  thread-like  growths. 
These  pseudo-mycelial  threads,  as  they  are  called,  indicate 
a  certain  relationship  to  the  group  of  moulds.  The  yeast 
cells  by  their  size  and  by  the  presence  of  budding  cells  can 
be  readily  distinguished  from  other  microscopic  forms. 
They  are  readily  stained  by  simple  anilin  dyes  and  by 
Gram's  method. 

When  actively  multiplying,  the  contents  of  the  yeast- 
cells  appear  perfectly  homogeneous  but  later  on  granules 
of  various  size  appear  and  surround  small,  clear  portions 
or  vacuoles.  The  contents  of  the  cell  on  contact  with 
iodine  take  on  a  brownish  violet  color  which  disappears  on 
heating  and  reappears  on  cooling.  This  is  due  to  the  pres- 
ence of  a  starch-like  or  glycogen  compound.  Occasionally 
a  gelatinous  envelope  or  capsule  may  form. 

Yeast  colonies,  when  they  develop  on  a  gelatin  plate, 
can  be  readily  recognized  even  with  a  low  power.  Owing 
to  the  large  size  of  the  cells  the  colony  has  a  peculiar, 
coarsely  granular  appearance.  They  do  not  liquefy  gelatin, 
although  they  do  contain  within  their  cells  a  proteolytic 
ferment. 

It  is  customary  to  divide  the  yeasts  into  two  groups 
according  as  to  whether  they  form  spores  or  not.  The 
;spore  producing  yeasts  are  designated  as  saccharomyces, 
whereas  those  forms  that  do  not  produce  spores  are  usually 
brought  together  under  the  group  name  torula. 

The   saccharomyces   as   indicated   above   give   rise   to 

*  endogenous  spores.     Usually  several  of  these  are  formed 

within  a  cell  as  shown  in  Fig.  55.     The  spores  can  be  double 

;stained  like  those  of  bacteria  or  like  the  tubercle  bacillus. 


YEASTS.  387 

.Most  of  the  saccharomyces  can  give  rise  to  alcohol  and  are, 
therefore,  of  great  industrial  importance.  Certain  species 
induce  the  alcoholic  fermentation  at  a  temperature  of 
14  to  18°  and  are  known  as  the  top  or  upper  yeasts,  whereas 
other  forms  are  active  at  a  lower  temperature,  4  to  10°,  and 
are  hence  commonly  designated  as  bottom  or  lower  yeasts. 


FIG.  55.    Feast  cells  with  spores  (Hansen). 

The  common  Saccharomyces  cerevisias  is  a  typical  upper 
yeast.  It  is  used  in  brewing  and  in  baking.  The  ordinary 
compressed  yeast  contains  this  organism  mixed  with  more 
or  less  starch.  It  forms  large,  round  or  oval  cells.  m  Not 
infrequently,  in  brewing,  this  organism  is  contaminated 
with  other  yeasts  or  even  with  bacteria,  and  the  products 
elaborated  by  these  foreign  organisms  may  greatly  alter 
the  composition  and  general  characteristics  of  the  beer. 
As  a  result  of  the  studies  of  Pasteur  and  of  Hansen,  on  these 
so-called  diseases  of  beer,  it  has  become  customary  in  large 
establishments  to  employ  only  pure  cultures  of  yeasts. 

The  most  common  cause  of  the  spontaneous  fermenta- 
tion of  grape  and  other  fruit  juice  is  the  Saccharomyces 
ellipsoideus.  Secondary  fermentations  or  ' '  diseases  "  may 
occur  in  wine  as  in  beer.  A  group  of  species  which  fre- 
quently show  sausage-like  cells  are  known  as  the  S. 
Pastorianus. 

The  torula  group  contains  the  so-called  wild  yeasts. 
These  rarely  give  rise  to  thread-like  forms,  and  never  pro- 
duce spores.  As  a  rule,  they  cannot  produce  more  than 


388  BACTERIOLOGY. 

about  one  per  cent,  of  alcohol,  and  some  species  are  wholly 
unable  to  ferment  sugar.  A  considerable  number  of  species 
have  been  described,  and  among1  these  may  be  especially 
mentioned  several  forms  which  are  widely  distributed  in 
the  air.  The  so-called  red-yeast  is  not  one  species  but 
rather  a  group  name  for  a  number  of  species  or  varieties. 
Red  and  white  torulae  are  frequently  deposited  from  the  air 
on  gelatin  plates.  Occasionally  a  black  yeast  is  met  with. 

Until  very  recently  the  yeasts  have  been  considered  as 
being  wholly  non-pathogenic.  When  introduced  in  large 
quantity  they  may  induce  a  catarrhal  condition  in  the 
stomach  and  in  the  intestines.  When  the  ordinary  yeasts 
are  injected  subcutaneously  apparently  no  bad  results  fol- 
low. Rabinowitsch,  however,  isolated  seven  varieties  of 
pathogenic  yeasts.  Monilia  Candida,  the  cause  of  thrush 
in  children,  is  a  yeast-like  organism  which  is  very  patho- 
genic for  mice  and  rabbits.  Mice  were  very  susceptible; 
rabbits  less  so,  and  guinea-pigs  were  refractory  to  the 
several  yeasts.  Gram's  method  can  be  used  to  advant- 
age for  the  detection  of  the  yeast  cells  within  the  animal 
body. 

Recently  yeast-like  forms  have  been  described  as  oc- 
curring in  certain  malignant  new  growths,  such  as  sarcoma 
and  cancer.  They  are  presumed  to  be  identical  with  the 
so-called  inclusion  cells. 

Moulds. 


This  group  of  organisms,  known  as  thread  fungi  or 
hyphomycetes,  cannot  be  defined  with  the  same  degree  of 
precision  as  is  the  case  with  the  bacteria  and  the  yeasts. 
It  includes  a  large  number  of  plants  which  differ  greatly  in 
form,  size,  shape  and  color.  As  a  rule  the  vegetative  form 
consists  of  threads  or  filaments  which  intertwine  or  inter- 


MOULDS.  389 

lace,  giving  rise  to  a  felt  or  cotton-like  growth  known  as 
the  mycelium. 

The  individual  filaments  or  threads  which  go  to  make  _ 
up  the  mycelium  are  known  as  mycelial  threads  or  as 
hyphce.  While  the  bacterial  cell  is  usually  about  1  p.  in 
width  the  mycelial  thread  will  vary  from  2-5-7  y.  in  width. 
In  other  words,  the  width  of  the  mycelial  thread  is  greater 
than  that  of  bacteria,  and  may  be  the  same  as  that  of  the 
yeasts. 

The  threads  may  be  very  short,  clinging  to  the  surface 
on  which  the  growth  is  developing;  or,  they  may  be  very 
long,  so  that  the  resulting  mycelium  may  have  a  height  of 
^-1  inch  or  more.  The  color  of  the  mycelium  will  vary  ' 
considerably  in  the  different  species.  It  may  be  pure  white, 
yellow,  green,  pink  or  black. 

Many  of  these  organisms,  especially  the  true  moulds, 
at  a  certain  stage  in  their  development  give  rise  to  repro- 
ductive cells,  and  eventually  spores  or  conidia  form. 
Usually  a  stalk  or  thread  known  as  the  fruit-hypha  rises 
upward  and  bears  on  its  end  the  fruit  organ.  The  latter  is 
so  characteristic  for  the  different  groups  or  families  that  it 
is  utilized  as  the  basis  of  a  natural  classification.  It  is 
thus  possible  to  divide  the  large  group  of  moulds  into  well 
defined  genera  and  even  species.  In  certain  forms,  inter- 
mediate between  the  true  moulds  and  the  bacteria,  the  fruit 
organs  are  wanting  or  are  imperfect,  and,  in  such  cases, 
the  classification  is  based  on  general  characteristics  such 
as  habitat,  size,  cultural  properties,  etc! 

In  general,  the  true  moulds  can  be  divided  into  two 
groups  according  as  the  spores  produced  are  contained  in 
a  sac  or  sporangium,  or  are  free  and  arranged  in  rows  on 
the  ends  of  modified  hyphas. 

A  large  number  of  families  or  groups  of  moulds  are  met 
with  accidentally  in  the  ordinary  routine  bacteriological 
work.  The  most  important  of  these  are  the  Mucor,  Asper- 
gillus,  Penicillium  and  Oidium. 


390 


BACTERIOLOGY. 


The  mucor  group  is  characterized  by  the  presence  of 
spherical  sacs  or  sporangia  on  the  ends  of  the  vertical  fruit 
hyphas.  The  contents  of  these  sacs  are  at  first  homogen- 
eous but,  eventually,  differentiation  into  round  or  oval 
spores  results.  When  fully  ripe  the  sac  bursts  and  the 
spores  are  set  free.  The  end  of  the  fruit  hypha  is  enlarged 
or  club  shaped  and  projects  into  the  sporangium,  forming 
what  is  known  as  the  columella  *(Fig.  56  a).  When  the  spor- 
angium bursts  the  membrane  disappears  or  dissolves  with 
the  exception  of  a  small  ring-  around  the  base  of  the  colum- 
ella. 


C 


FIG.  56.  Fruit-organs  of  moulds,  after  Lehmann.  A. — Sporangium  of  mucor,  rilled 
with  spores,  showing  columella;  B — Aspergillus  with  sterigmse  and  spores;  C— Penicillium 
with  basidia  and  spores. 

The  mucors  also  give  rise  to  zygospores  which  result 
by  the  union  of  the  ends  of  two  mycelial  threads.  The 
mycelial  thread  up  to  the  fruiting  stage  is  unseptate,  and 
represents,  therefore,  a  continuous  tube.  When  grown 
in  liquids  the  thread  character  gives  place  to  yeast-like 
cells.  3  or  4  species  of  this  group  are  capable  of  inducing 
experimental  pathogenic  effects.  In  very  rare  instances 
mucor  mycoses  have  been  met  with  in  man. 

The  aspergillus  group  has  no  sporangia  but,  instead,  the 
ends  of  the  fruit-hyphae  are  enlarged  or  club-shaped,  the 
enlarged  end,  or  columella,  is  covered  with  radially  ar- 
ranged, minute  bottle-shaped  bodies— the  intermediate 
spore  bearers  or  sterigmce — from  which  chains  or  rows  of 
spores  extend  outward.  Additional  fruit  organs,  perithecia 


MOULDS.  391 

and  sclerotia,  are  sometimes  present.  The  mycelial  threads 
branch  freely,  but  the  fruit  hyphas  are  not  divided  into 
cells  (Fig.  56  b). 

The  penicillium  has  fruit  organs  resembling  somewhat 
those  of  the  preceding  group.  The  fruit  hyphae,  however, 
are  septate  and  divide,  and  each  branch  ends  in  a  charac- 
teristic brush-like  form.  The  end  of  each  branch  is  not  en- 
larged as  in  the  case  of  the  mucor  or  aspergillus  type,  but 
like  the  latter  it  is  covered  by  a  number  of  bottle-shaped 
intermediate  spore-bearers,  known  as  basidia.  Each  of 
these  in  turn  bears  a  chain  or  row  of  spores  or  conidia.  As 
many  as  8  spores  may  be  held  in  a  row  on  the  end  of  the 
bottle-shaped  basidium  (Fig.  56  c). 

The  oidium  does  not  possess  a  definite  fruit  organ  such 
as  has  been  described  in  connection  with  the  preceding 
forms.  The  mycelial  threads  branch  and  are  usually  made 
up  of  short  thick  cells.  From  the  ends  of  the  thread  or 
from  the  ends  of  the  cells,  chains  of  large,  oval  conidia  are 
given  off.  On  sugar  media  it  will  slowly  give  rise  to  alco- 
hol. The  odor  of  limburger  cheese  may  be  largely  due 
to  the  Oidium  lactis. 

The  moulds,  to  a  certain  extent,  induce  fermentative 
decompositions(pp.  93,  97).  They  are  of  especial  interest  as 
the  causes  of  certain  diseases  in  plants,  in  lower  animals 
and  in  man.  At  the  beginning  of  the  century,  certain 
botanists  held  that  the  blight  of  wheat  and  of  other  plants 
was  due  to  a  microscopic  parasite.  The  first  actual  dem- 
onstration of  the  relation  of  a  fungus  to  a  diseased  organ- 
ism was  supplied  in  1837  by  Bassi,  who  showed  that  the 
silk-worm  disease,  known  as  muscardine,  was  due  to  a  mould 
— Botrytis  Bassiana.  In  the  same  year  the  relation  of  the 
yeast  plant  to  alcoholic  fermentation  was  for  the  first  time 
clearly  indicated.  The  possibility  of  microscopic  moulds 


392  BACTERIOLOGY. 

being  the  cause  of  disease  in  plants  and  animals  lead  the 
investigators  of  that  period  to  make  thorough  studies  of 
such  affections.  This  led  to  the  discovery  during  the  fourth 
decade  of  a  large  number  of  moulds  which  were  shown  to  be 
pathogenic  for  man,  animals  and  plants. 

Several  of  these  fungous  diseases  may  be  briefly  men- 
tioned. The  blight  on  potato  leaves  is  due  to  the  Perono- 
spora  infestans.  The  mildew  of  grapes  and  other  plants  is 
due  to  various  species  of  Oidium.  The  ergot  of  rye  and 
other  grasses  is  due  to  the  Claviceps  purpurea.  The  smut  on 
corn  and  other  plants  is  due  to  several  species  of  the  fun- 
gus Ustilago.  Bunt,  the  blight  which  attacks  the  grain  of 
wheat  and  spelt  forming  a  black  fetid  powder  or  mass  of 
spores,  is  due  to  another  mould,  the  Tilletia.  The  rust  or 
mildew  on  grains  is  due  to  various  species  of  the  Puccinia. 

The  parasitic  mould  which  can  be  seen  on  dead  flies  is 
known  as  the  Empusa  muscce.  •  The  silk- worm  disease,  mus- 
cardine,  as  stated  above  is  due  to  the  Botrytis  Bassiana. 

The  skin  affection  in  man,  known  as  Herpes,  is  due  to 
a  group  of  fungi,  commonly  designated  as  Tricophyton  ton- 
surans,  which,  by  some  is  placed  in  the  Botrytis  group.  The 
fungus  of  thrush  in  children  is  known  as  the  Monilia  Candida 
and  by  some  is  placed  in  the  Oidium  group.  Favus  is  a  skin 
disease  of  man  due  to  the  Achorion  Schdnleinii.  Similar  af- 
fections are  known  to  exist  among  birds,  mice  and  other 
animals.  Another  cutaneous  disease,  pityriasis  is  ascribed 
to  the  fungus  Microsporon  furfur  which,  however,  has  not  as 
yet  been  successfully  cultivated. 

Streptotrices. 

Under  this  name  are  included  certain  saprophytic,  and, 
at  times,  pathogenic  organisms  which  show  some  resem- 
blance to  the  moulds  as  well  as  to  the  bacteria.  In  other 
words,  they  are  to  be  considered  as  intermediate  forms. 


STREPTOTRICES.  393 

Like  the  moulds,  they  consist  of  cylindrical  cells  which 
branch  dichotomously  and  form  radially  arranged  masses 
or  mycelia.  Moreover,  f ruit-hyphas  develop  and  bear  chains 
of  roundish  spores  or  conidia.  Many  of  these  forms  when 
growing  in  the  animal  body  or  in  pure  culture  form  hard 
masses  which  have  a  radial  structure. 

The  resemblance  to  the  bacteria  is  especially  evident 
under  the  microscope.  The  filaments,  unlike  those  of  the 
moulds,  are  extremely  narrow — about  the  same  width  as 
the  average  bacterial  cell.  These  threads  branch  freely  and 
are  perfectly  homogeneous,  without  any  transverse  division 
into  cells  as  in  the  case  of  the  moulds.  As  the  threads  be- 
come old  they  may  break  up  into  rod-,  or  coccus-like  forms 
not  unlike  bacteria.  Even  spiral  forms  may  thus  result. 
This  fragmentation  of  the  filament  is  to  be  distinguished 
from  the  segmentation  process  whereby  the  round  spores 
or  conidia  form  on  the  ends  of  the  fruit-hyphae. 

The  spores  of  these  organisms  have  not  the  resistance 
of  those  of  bacteria.  Thus,  in  the  case  of  the  actinomyces 
they  are  destroyed  in  5  minutes  at  75°.  Moreover,  they  are 
stained  readily  by  the  simple  anilin  dyes  and  by  Gram's 
method. 

The  diphtheria  and  tubercle  bacilli  undoubtedly  belong 
to  this -group,  although  their  predominant  characteristics 
are  those  of  bacteria.  Nevertheless,  branching  forms  have 
been  observed  in  both  germs.  The  fragmentation  of  the 
cells  into  rod-,  or  coccus-like  forms  is  analogous  to  that  of 
the  true  streptothrix.  The  club-shaped  rods  are  involution 
forms  similar  to  those  observed  in  actinomyces.  More- 
over, the  necrotic  action  or  the  production  of  new  growths 
by  these  bacilli  differs  in  no  wise  pathologically  from  sim- 
ilar changes  induced  by  the  true  streptothrix. 

Under  the  term  streptothrix  are  to  be  classed:  the 
fungus  of  lumpy- jaw,  of  which  there  are  perhaps  several 
species;  that  of  farcy  in  cattle,  and  that  of  Madura-foot 
in  man.  Moreover,  many  forms  of  pseudo-tuberculosis  in 


394  BACTERIOLOGY. 

man  and  animals  are  due  to  these  organisms.  Other 
pathogenic  species  have  been  found,  though  but  rarely,  in 
man  and  in  the  lower  animals.  Many  of  the  streptotrices 
are  found  in  the  air  and  in  water.  The  members  of  this 
group,  like  the  moulds  and  yeasts,  are  stained  by  Gram's 
method. 

Laboratory  work. — The  student  will  inoculate  ordinary  gelatin,  or 
better  glucose  gelatin,  with  the  several  yeasts.  The  Saccharomyces 
cerevisiae,  or  the  white  yeast  is  to  be  poured  into  Petri  dishes,  while 
the  red  •  or  black  yeasts  are  to  be  used  for  making  Esmarch  roll- 
tubes. 

Ordinary  baker's  or  brewer's  yeast  should  be  examined  in  the 
hanging-drop  and  in  stained  preparations.  If  compressed  yeast  is 
employed  it  will  be  well  to  distinguish  the  budding  yeast-cells  from 
the  very  large  starch  grains  that  are  present. 

The  yeasts  are  fixed  on  the  cover-glass  and  stained  in  the  same 
way  as  bacteria. 

The  moulds  grow  best  on  slightly  acid  media.  They  can,  there- 
fore, be  grown  to  advantage  on  the  surface  of  potatoes  or  on  moist 
bread.  The  addition  of  about  2  per  cent,  of  cane-sugar  or  of  glycerin 
to  agar  renders  this  a  very  good  medium.  The  Oidium  lactis  should 
be  grown  on  gelatin  plates. 

Preparation  of   Bread-flasks. 


A  bread  powder  is  usually  first  prepared.  For  this 
purpose  the  bread  is  cut  up  into  slices  and  heated  in  a 
dry-heat  oven  till  over-toasted.  It  is  then  finely  crushed 
and  preserved  in  a  bottle.  The  dry  powder  can  be  kept 
indefinitely  in  a  well  stoppered  bottle. 

Six  small,  30-50  c.c.  Erlenmeyer  flasks'  are  cleaned, 
plugged  and  sterilized.  The  dry,  powdered  bread  is  filled 
into  each  flask  to  a  depth  of  about  i  inch,  and  water  is  then 
added  so  as  to  render  the  mass  thoroughly  moist.  The 
bread-flasks  are  now  steamed  for  half  an  hour  on  each  of 
three  successive  days. 


PREPARATION  OF   BREAD-FLASKS.  395 

The  sterile  bread-flasks  are  inoculated  with  the  follow- 
ing' moulds: 

Penicillium  glaucum.  Aspergillus  niger. 

Mucor  corymbifer.  Asperglllus  flavescens. 

Mucor  rhizopodiformis.  Aspergillus  fumigatus. 

All  these  flasks,  except  the  first,  should  be  placed  in 
the  incubator  at  about  37°,  for  24  to  36  hours.  They  should 
then  be  examined  for  the  characteristic  fruit-organs  and 
for  spores.  A  portion  of  the  growth  for  this  purpose  is 
transferred  to  a  watch-glass  containing  some  50  per  cent, 
alcohol,  to  which  a  drop  or  two  of  ammonium  hydrate  has 
been  added.  When  the  growth  becomes  moist,  it  should  be 
transferred  to  a  drop  of  glycerin  on  a  slide.  The  specimen 
is  thoroughly  and  carefully  teased  with  needles  or  pins.  It 
is  finally  covered  with  a  glass-slip  and  examined  with  the 
No.  7  objective. 

If  the  specimen  is  satisfactory  it  may  be  made  perma- 
nent by  placing  a  ring  of  asphalt,  with  the  aid  of  a  turn- 
table, around  the  edge  of  the  cover-glass.  Although  the 
moulds  stain  readily  by  the  simple  and  by  Gram's  method, 
it  is  not  advisable  to  prepare  dried  specimens  owing  to  the 
alteration  which  results  during  desiccation. 

In  this  laboratory  it  is  customary  to  study  the  yeasts 
and  moulds  at  the  close  of  the  work  on  non-pathogenic 
bacteria.  The  parasitic  skin  moulds  and  the  streptothrix 
forms  are  studied  in  connection  with  the  pathogenic 
bacteria  (see  p.  316). 


Saccharomyces  Cerevisiae. 

ORIGIN. — Beer  or  bakers'  yeast;  at  times  in  the  air. 
COLOR.— White. 

FORM. — Cells  spherical  or  egg-shaped  8-10  p.  broad.  The 
cells  are  colorless  and  when  actively  growing  have  a  homo- 
geneous protoplasm.  Later,  granules  and  vacuoles  develop. 
Owing  to  a  gelatinous  exudate  it  may  form  zoogleal  masses. 
The  cells  may  be  single  or  may  have  several  buds;  at  times 
long  branching  forms  may  be  found,  especially  if  the  tem- 
perature is  about  30°. 

MOTILITY.  — None. 

SPORULATION. — Usually  several  spores  form.  Can  be 
double  stained.  Spores  develop  between  11  and  37°. 

ANILIN  DYES. — Stain  readily;  so  does  Gram's  method. 

GROWTH.— Thick  white  growth,  especially  abundant  on 
glucose  media  and  in  wort. 

Gelatin  plates. — Small,  opaque,  white,  circular  colonies  which  are 
very  coarsely  granular  and  slimy. 

Stab  culture, — The  growth  is  confined  to  the  upper  portion  of  the 
tube  and  spreads  over  the  surface.  It  is  thick  and  opaque  white. 

Streak  culture. — On  agar  and  on  potato  it  forms  a  thick,  somewhat 
raised  white  growth. 

TEMPERATURE. — As  an  "upper  yeast"  the  most  rapid 
fermentation  takes  place  between  14  and  18°. 

BEHAVIOR  TO  GELATIN. — Does  not  liquefy  gelatin. 

AEROGENESIS.— It  gives  rise  to  a  ferment  (invertin)  which 
changes  cane-sugar  into  glucose.  The  latter  is  then 
changed  by  another  ferment,  zymase,  to  carbonic  acid  and 
alcohol  (4-6  per  cent.).  It  does  not  ferment  lactose. 

PATHOGENESIS.— It  has  no  effect  on  animals.  A  large 
amount  may  produce  a  catarrhal  condition  in  the  alimentary 

tract. 

396 


DRAWINGS.  397 


Saccharomyces  Glutinis. 

RED   YEAST. 

ORIGIN. — Very  common  in  the  air,  from  which  several 
distinct  kinds  of  red  yeast  have  been  obtained. 

COLOR. — Red  or  pink. 

FORM. — Round  or  oval  cells  with  granular  protoplasm 
which  stains  irregularly.     Cells  single  or  in  pairs,  budding-. 
MOTILITY.  — None. 
SPORULATION.  —  None. 

ANILIN  DYES. — It  stains  readily,  also  by  Gram's  method. 
GROWTH. — Abundant,  though  somewhat  slow. 

Gelatin  plates. — Colonies  are  small,  round,  elevated,  moist  and 
pink-colored  and  coarsely  granular. 

Stab  culture.— Growth  absent  from  the  lower  part  of  the  tube.  It 
spreads  slowly  over  the  surface,  forming-  a  thick,  moist,  bright  red 
covering. 

Streak  culture. — On  agar,  it  develops  in  a  few  days  as  a  thick, 
slimy,  spreading,  pink-colored  growth.  On  potato,  it  forms  the  same 
pigment. 

TEMPERATURE. — Grows  best  at  ordinary  temperature. 
BEHAVIOR  TO  GELATIN. — Does  not  liquefy. 
AEROGENESIS. — Does  not  change  glucose  to  alcohol. 
PATHOGENESIS. — No  effect  on  animals. 

A  white  yeast  is  frequently  deposited  from  the  air. 
The  cells  are  usually  smaller  than  those  of  the  red  yeast. 

A  black  yeast,  8.  niger,  forms  a  brownish  or  black 
growth.  The  size  of  the  cell  is  about  the  same  as  that  of 

the  red  yeast.     It  grows  very  slowly. 

398 


DRAWINGS.  399 


Oidium  Lactis. 

ORIGIN. — Almost  invariably  present  in  milk  and  in  but- 
ter; also  in  sugar  solutions  of  various  kinds,  and  in  brewer's 
yeast. 

FORM. — A  delicate,  white  mycelium  of  forked,  wavy 
threads.  No  special  fruit-organ  is  present.  The  conidia 
or  spores  are  very  irregular  in  size  and  form;  they  may  be 
oblong,  round  or  oval. 

ANILIN  DYES. — React  readily.  The  specimen  shrinks  on 
drying. 

GROWTH. — Is  rapid,  especially  on  acid  media. 

Gelatin  plates. — Delicate  white  stars  form,  which  rapidly  enlarge, 
and  spread  on  the  surface  as  dry,  flat,  whitish  masses.  Under  the 
microscope  the  colonies  show  radiating,  branched  hyphae  and  chains 
of  conidia. 

Stab  culture. — Growth  takes  place  along-  the  entire  line  of  inocu- 
lation, and  is  most  abundant  at  or  near  the  surface.  A  branching- 
network  of  threads  extends  outward  into  the  solid  gelatin.  On  the 
surface  a  grayish  white,  dry,  low  growth  forms.  In  old  cultures  only 
the  upper  layer  of  gelatin  shows  the  radiating  lines. 

Streak  culture.— On  agar,  it  forms  a  grayish  white,  thin  growth. 
Milk. — Growth  occurs  without  altering  the  composition. 

TEMPERATURE. — Grows  best  at  ordinary  temperature. 
It  can  grow  in  the  incubator. 

BEHAVIOR  TO  GELATIN. — Does  not  liquefy. 
PATHOGENESIS. — It  has  no  effect   on  animals.     A  some- 
what  similar  organism,   the   Oidium    Tuckeri,  produces  a 

vine  disease. 

400 


DRAWINGS.  401 


Monilia  Candida,  Robin  (1847). 

THRUSH  FUNGUS;  OIDIUM  ALBICANS,  SACCHAROMYCES  ALBICANS; 
MUGUET  (Fr.);  SOORPILZ  (Germ.). 

ORIGIN. — Found  in  thrush  on  the  mucous  membrane  in 
the  mouths  of  infants  and  of  grown  persons;  also  in  air,  in 
milk,  in  barns;  occurs  as  a  white  growth  on  cow-dung.  It 
may  occasionally  be  found  on  mucous  membrane  other  than 
that  of  the  mouth. 

FORM. — Occupies  an  intermediate  position  between  the 
moulds  and  yeasts.  On  gelatin  plates  and  on  sugar  media 
it  forms  yeast-like  cells  (conidia),  whereas  in  the  deeper 
part  of  a  stab  culture  it  forms  mycelial  threads.  The 
mycelial  threads  are  developed  best  in  the  absence  of  sugar 
and  at  a  high  temperature. 

ANILIN    DYES. — Stain  readily. 

GROWTH. — Is  rapid  and  abundant. 

Plates. — Snow-white,  coarsely  granular  colonies  form  on  gelatin 
plates  and  no  liquefaction  takes  place. 

Stab  culture.— In  gelatin  show  a  slight  growth  along  the  line  of  in- 
oculation, while  on  the  surface  a  milk-white,  thick  mass  forms. 

Streak  culture. — On  agar,  forms  a  glistening,  moist,  thick,  white 
growth.  On  potato,  it  grows  rapidly  as  a  thick,  white,  yeast-like  mass. 

OXYGEN  REQUIREMENTS. — It  is  an  aerobe. 

TEMPERATURE. — Grows, at  the  ordinary  temperature;  also 
in  the  incubator  at  40°. 

BEHAVIOR  TO  GELATIN. — It  does  not  liquefy. 

PATHOGENESIS. — Intravenous  injection  in  rabbits  pro- 
duces death  in  1-2  days;  with  very  small  doses  death  may 
not  result  for  a  week  or  two.  The  internal  organs  are 
sometimes  permeated  with  a  growth  of  long  mycelial 
threads.  Under  rare  conditions  it  has  been  found  in  the 
internal  organs  of  man.  When  inoculated  into  the  throat 
of  doves  and  chickens  it  produces  a  typical  thrush-mem- 
brane. Subcutaneous  application  of  a  few  mg.  will  produce 
death  in  white  mice  in  1-3  days.  It  has  no  effect  on  guinea- 
pigs.  The  identity  of  Monilia  Candida  with  thrush  cannot  be 
said  to  be  fully  established. 

It  ferments  cane-sugar  and  maltose  without  previous 
inversion,  and  in  time  may  give  rise  to  about  5  per  cent,  of 
alcohol. 

402 


DRAWINGS.  403 


Mucor  Corymbifer,  Lichtheim. 

V 

ORIGIN. — Is  of  rare  occurrence,  and  was  found  as  a  con- 
tamination on  bread- gelatin  plates  and  on  white  bread 
which  was  kept  at  the  body  temperature.  It  has  been 
found  in  the  ear-passages  of  man.  It  is  probable  that  this 
same  species  has  been  found  in  a  case  of  generalized 
mycosis. 

COLOR. — The  mycelium,  spores  and  sporangia  are  color- 
less. 

MYCELIUM. — Loose,  wavy,  branching,  slender  mycelial 
threads  which  form  a  white  cotton-like  mass  an  inch  or 
more  in  height. 

FRUIT-ORGANS. — The  fruit-hyphas  branch  forming  clusters 
or  corymbs  which  terminate  in  spherical  or  pear-shaped 
sporangia.  Within  these  are  the  colorless,  oval  or  elongated 
spores,  which  are  about  3  P.  long  and  2  //  wide. 

GROWTH. — Rapid  and  extensive. 

Bread-flasks — In  the  incubator  it  forms  a  white,  elevated,  cotton- 
like  growth  which  soon  fills  the  flask.  On  potato  a  similar  cotton-like, 
tall  growth  develops. 

TEMPERATURE. — Grows  slowly  at  the  ordinary  tempera- 
ture; best  at  37°. 

PATHOGENESIS. — Intravenous  injection  of  the  spores  into 
rabbits  produces  death  in  3  to  4  days.  The  kidneys, 
mesenteric  glands  and  Peyer's  patches  contain  mycelial 
masses.  The  Peyer's  patches  are  swollen  and  ulcerated. 
Intraperitoneal  injections  produce  the  same  results.  Dogs 

are  immune. 

404 


DRAWINGS'.  405 


Mucor  Rhizopodiformis,  Lichtheim. 

ORIGIN.— White  bread  kept  at  37°. 

COLOR. — At  first  white,  but  later  becomes  grayish. 

MYCELIUM. — The  mycelial  threads  are  colorless,  later  on 
brownish,  and  thicker  than  those  of  the  preceding-  mucor. 
They  are  not  jointed  or  divided.  At  first  the  growth  is 
white,  then  becomes  grayish. 

FRUIT-ORGANS. — The  fruit  hyphae  occur  in  groups  or 
bunches,  which  adhere  to  the  nutrient  medium  by  means  of 
special  root  tufts.  The  larg-e  dark  sporangia  on  the  ends 
of  the  hyphae  contain  colorless  rounded  spores  which  are 
larg-er  than  those  of  the  preceding-  org-anism  (5-6  ^). 

GROWTH. — Is  rapid  and  has  a  pleasant  odor. 

Gelatin  plates. — Development  is  best  when  the  gelatin  is  made 
with  bread  infusion.  It  forms  a  coarse  grayish-black  mass  which 
liquefies  the  gelatin. 

Bread  flasks.  —The  growth  is  lower  than  that  of  M.  corymbifer, 
and  is  grayish,  owing  to  the  dark  colored  sporangia.  An  ethereal  or 
aromatic  odor  is  present. 

TEMPERATURE. — Slow  growth  at  12  to  15°;  develops  best 
at  37°. 

BEHAVIOR  TO  GELATIN. — Liquefies. 

PATHOGENESIS. — It  has  a  similar  effect  as  M.  corymbifer, 
but  is  more  pathog-enic. 

The  mucors  are  characterized  by  marked  fermentative 
powers.  They  convert  dextrose  and  maltose  into  alcohol. 
Only  one  species,  however,  can  invert  cane-sug-ar.  Some 
species  may  produce%7-8  per  cent,  of  alcohol  and  many  of 
them  give  rise  to  diastatic  ferments.  The  most  common 
species  is  the  M.  mucedo  which  is  relatively  frequent  on  the 
excreta  of  herbivorous  animals.  The  M.  racemosus  is  like- 
wise common  and  its  spores  are  found  in  the  air. 

406 


DRAWINGS.  407 


Aspergillus  Niger,  Van  Tieghem. 

ORIGIN. — In  putrid  substances;  in  the  lungs  of  birds. 

COLOR. — Black  or  dark  brown. 

MYCELIUM. — This  is  low  and  at  first  white,  then  brown- 
ish or  black. 

FRUIT-ORGANS. — The  fruit  hyphse  are  spherical,  or  flask-, 
or  club-shaped  at  the  end,  and  this  enlargement  is  covered 
with  radially  arranged,  minute  bottle-shaped  bodies — the 
intermediate  spore  bearers  or  sterigmce — from  which  rows  of 
spores  extend.  The  sterigmae  are  divided.  The  spores 
are  black  or  brownish  and  spherical,  and  are  3-5  ft  in 
diameter. 

GROWTH.  — Slow. 

Bread  flasks. — It  forms  a  low  growth  which  becomes  very  black. 

TEMPERATURE. — The  optimum  is  about  35°. 

PATHOGENESIS. — Intravenous  injection  of  spores  in  rab- 
bits is  not  followed  by  as  malignant  results  as  with  the 
next  two  forms.  It  gives  rise  to  diastatic,  inverting  and 
other  ferments. 

Aspergillus  Flavescens,  Wreden. 

ORIGIN. — White  bread. 

COLOR. — At  first  whitish,  eventually  pale  yellow  or  yel- 
lowish green. 

MYCELIUM. — The  mycelial  threads  and  spores  are 
smaller  than  those  of  A.  niger. 

FRUIT-ORGANS. — The  club-shaped  ends  of  the  fruit 
hyphae  are  covered  with  sterigmae,  from  which  extend 
rows  of  spores,  as  in  A.  niger.  The  spores  are  yellowish 
or  brownish  in  color  and  are  5-7  ft  in  diameter. 

GROWTH.  — Rapid. 

Bread  flasks. — Grows  best  on  bread  where  it  forms  a  yellowish, 
low  growth. 

TEMPERATURE. — The  optimum  is  about  28°,  but  it  grows 
well  in  the  incubator. 

PATHOGENESIS. — It  is  more  pathogenic  than  A.  niger, 
and  less  than  A.  fumigatus. 

408 


DRAWINGS. 


409 


Aspergillus  Fumigatus,  Lichtheim. 

ORIGIN.— White  bread;  in  the  air  passages  and  lungs  of 
birds;  also  met  with  in  man. 

COLOR. — Greenish  or  bluish  green  growth,  resembling 
very  much  that  of  penicillium. 

MYCELIUM. — About  the  same  as  that  of  the  preceding. 

FRUIT-ORGANS. — Like  the  preceding,  but  the  spores  are 
only  about  one-half  as  large  and  are  usually  colorless. 
The  sterigmse  do  not  divide. 

GROWTH. — Is  best  on  bread  and  is  rapid. 

Bread  llasks. — The  growth  is  low  and  at  first  bluish  green,  but 
when  old  is  grayish  green  and  resembles  that  of  Penicillium. 

TEMPERATURE. — The  optimum  is  37-40°.  Tt  can  grow  at 
the  ordinary  temperature,  but  not  below  15°. 

PATHOGENESIS. — Intravenous  injections  of  millions  of 
spores  in  rabbits  and  dogs  produced  death  in  a  few  days. 
Mycelia  were  found  in  the  kidneys,  heart  and  other  muscles,. 

and  occasionally  in  the  liver. 

•  %._ 

Infection  of  doves  and  other  birds  by  inhalation  of  the 
spores  produces  a  pneumonic  or  pseudo-tuberculous  disease. 
Natural  affections  of  this  kind  are  frequently  observed 
among  birds.  Occasionally,  they  are  met  with  in  horses 
and  in  cattle,  and  at  times  in  man. 

In  mycoses  of  man,  the  lungs,  ears,  eyes  or  nose  are 
subject  to  invasion. 

The  Japanese  utilize  the  growing  A.  Oryzae  as  a  diasta- 
tic  ferment,  like  malt.  It  converts  rice  grains  into  sugar 
and  dextrin.  This  liquid  is  then  subjected  to  fermentation 
and  yields  the  national  drink,  Sake,  which  contains  about 
14  per  cent,  of  alcohol.  Taka-diastase  is  a  ferment  derived 

from  an  aspergillus  like  that  mentioned. 

410 


DRAWINGS. 


411 


Penicillium    Glaucum. 

ORIGIN. — Widely  distributed  in  the  air,  water  and  soil. 
60  per  cent,  of  the  mould  contaminations  in  the  laboratory 
are  said  to  be  due  to  this  organism. 

COLOR. — Is  at  first  whitish,  then  becomes  bluish-green. 

MYCELIUM. — Consists  of  horizontally  arranged,  straight 
or  slightly  wavy,  jointed  mycelial  threads  from  which  the 
fruit  hyphas  rise  vertically. 

FRUIT-ORGANS. — The  ends  of  the  septate  fruit  hyphae  are 
forked,  and  are  covered  with  the  intermediate  spore  bear- 
ers, or  sterigmae,  also  sometimes  called  basidia.  Each  of 
these  in  turn  bears  a  row  of  8  spores  or  conidia,  so  that 
the  appearance  of  the  whole  is  that  of  a  brush.  The 
spores  are  about  3.5  P.  wide. 

GROWTH. — Is  rapid. 

Gelatin  plates.  —The  colonies  form  whitish  floccules  which  rapidly 
increase  in  size,  and  at  the  same  time  the  center  colors  green.  The 
gelatin  is  liquefied  quite  early.  A  low  objective  will  show  the  above 
characteristics  of  growth. 

Bread  flasks.— Show  a  low,  finely  flocculent  covering,  which  at 
first  is  white  but  soon  changes  to  a  distinct  green. 

TEMPERATURE.— The  optimum  temperature  is  from  22  to 
26°.  It  does  not  grow  at  the  temperature  of  the  body. 

BEHAVIOR  TO  GELATIN.  —  Slowly  liquefies. 

PATHOGENESIS. — It  has  no  effect  on  animals.  It  fre- 
quently develops  on  grapes  and  causes  a  marked  alteration 
in  wine.  It  gives  rise  to  inverting  and  to  diastatic  fer- 
ments. This  organism  is  said  to  be  used  in  the  preparation 

of  Roquefort  cheese. 

412 


DRAWINGS. 


Achorion  Schdnleinii,  Schonlein  (1839). 

THE  FUNGUS   OF   FAVUS. 

ORIGIN. — Found  in  the  scaly  accumulations  on  the  skin 
of  persons  afflicted  with  favus.  Similar,  if  not  identical 
organisms  occur  in  the  favus  of  the  dog-,  cat,  rabbit,  mouse, 
chicken,  etc.  It  is  closely  related  to  the  Oidium  lactis. 

FORM. — Apparently  belongs  to  the  moulds  that  produce 
oidia.  The  mycelium  when  actively  developing-,  consists  of 
stellate  threads  which  are  not  septate.  The  individual 
hyphae  vary  considerably  in  thickness  and  usually  fork. 
The  ends  are  swollen  and,  moreover,  peculiar  yellowish 
lateral  buds  or  corpuscles  are  seen.  When  the  culture  be- 
comes old  the  threads  divide  into  oval  elements  (oidia) 
which  remain  attached.  In  the  deeper  layers  of  the  culture 
medium,  moss-like  ramifications  are  found. 

FRUIT-ORGANS. — No  true  fruit-organs  observed,  but  on 
special  media,  as  on  blood-serum  at  30°,  conidia  or  spores 
are  said  to  form. 

ANILIN  DYES. — Stain  well;  so  does  Gram's  method. 

GROWTH. — Is  rather  slow  and  requires  a  week  or  more. 
It  is  at  first  grayish-white,  then  yellowish.  On  the  usual 
media  it  tends  to  grow  below  the  surface  and  only  a  feeble 
development  occurs  in  contact  with  the  air. 

Plates. — On  gelatin,  the  colonies  grow  slowly  and  form  whitish, 
stellate  masses  which  liquefy  the  gelatin.  No  conidia  present. 

Stab  culture. — Growth  is  very  poor  in  the  lower  part  of  the  gelatin 
tube.  On  the  surface  it  forms  a  white  covering-,  the  lower  side  of 
which  is  light-yellow.  On  potato  it  forms  thick  scales. 

Streak  culture. — On  agar  it  forms  a  closely  adherent,  dry,  folded, 
whitish  mass. 

TEMPERATURE. — Dies  out  at  the  ordinary  temperature. 
The  optimum  is  about  30°. 

BEHAVIORTO  GELATIN. — Slowly  liquefied;  becomes  reddish. 

PATHOGENESIS. — Inoculation  with  a  pure  culture  produces 
typical  favus  in  man.  It  is  usually  localized  on  the  scalp. 

The  favus  fungus  is  closely  related  to  that  of  Herpes 
tonsurans — the  Tricpphyton  tonsurans  (1845);  and  to  that  of 
Pityriasis  versicolor — the  Microsporon  furfur  (1846). 

In  order  to  isolate  the  organism  in  pure  culture  it  is 
advisable  to  crush  the  scales  in  a  sterile  mortar  with  ster- 
ile sand.  This  finely  divided  powder  is  then  used  to  make 
.dilution  cultures  on  ordinary  agar.  As  many  as  nine  dif- 
ferent varieties  of  Achorion  have  been  described. 

414 


DRAWINGS.  415 


Streptothrix  Actinomyces,  Bellinger  (1877). 

ACTINOMYCES   BOVIS;   RAY-FUNGUS;   STRAHLENPILZ  (Germ.). 

ORIGIN. — Occurs  in  actinomycosis  or  lumpy-jaw  in  cat- 
tle, hogs,  horses  and  in  man.  It  probably  leads  a  sapro- 
phytic  existence  on  plants,  etc. 

FORM. — It  gives  rise  to  nodules  which  consist  of  a 
whorl  of  mycelial-like,  multiple  branched  threads.  These 
radiate  outward  from  a  central  point  and  become  club- 
shaped.  In  pure  cultures  only  slender,  wavy  threads  are 
formed.  The  club-shaped  or  swollen  ends  which  are  usually 
present  in  tissues  are  lacking,  unless  the  organism  is  grow- 
ing deep  in  gelatin  or  in  blood-serum.  The  club-shaped 
ends  are  the  result  of  degenerative  changes.  In  other 
words,  they  are  involution  forms  due  to  a  gelatinization  of 
the  cell-wall. 

ANILIN  DYES. — It  stains  readily  with  carbolic  fuchsin; 
also  by  Gram's  method. 

GROWTH. — Develops  somewhat  slowly,  requiring  several 
days  in  the  incubator.  It  can,  .however,  grow  on  various 
media  even  at  the  ordinary  room  temperature.  Although 
usually  yellowish  it  may  take  on  a  brick-red  color. 

Streak  culture. — On  agar,  the  growth  begins  as  minute,  isolated 
colonies  which  slowly  enlarge,  forming  thick,  convex,  glistening,  yel- 
lowish, opaque  masses.  The  colonies  are  exceedingly  hard  and  for 
examination  should  be  crushed  between  two  glass  slides,  previously 
sterilized  by  passing  several  times  through  the  flame.  Cover-glass 
preparations  are  then  made  and  stained  in  the  usual  manner. 

In  bouillon  the  growth  develops  on  the  bottom  and  does  not  cloud 
the  liquid. 

OXYGEN  REQUIREMENTS. — Said  to  grow  best  in  the  ab- 
sence of  air,  but  grows  very  well  on  the  surface  of  agar. 

TEMPERATURE. — It  grows  best  at  or  near  that  of  the 
body. 

PATHOGENESIS. — In  rabbits,  intraperitoneal  injection  of 
the  pure  culture  is  said  to  produce  typical  actinomycotic 
nodules  on  the  peritoneum,  mesentery,  intestinal  walls,  etc. 

The  disease  is  recognized  by  the  presence  of  small  yel- 
lowish granules  in  the  pus  which  is  derived  from  the  tumors. 
The  granules  when  examined  with  a  No.  3  objective  will 
show  the  typical  stellate  growth,  the  hyphae  of  which  have 
club-shaped  ends. 

Infection  results  from  the  plant  food  on  which  the  or- 
ganism is  growing.  Several  varieties  of  actinomyces  have 
been  described;  can  be  distinguished  only  by  culture. 

416 


DRAWINGS.  417 


27 


Streptothrix  Madurse,    Vincent  (1894). 


ORIGIN. — Occurs  in  Madura  Foot,  or  Mycetoma,  a  disease  which  is 
endemic  in  India.  It  has  been  met  with  in  Italy,  Africa  and  several 
cases  have  been  studied  in  this  country.  The  discharge  in  some  cases 
contains  minute,  black,  gun-powder-like  grains;  in  others,  grayish  or 
yellowish  granules  are  present.  These  granules  are  due  to  mycelial 
growths  and  resemble  those  found  in  actinomycosis. 

FORM. — It  consists  of  slender,  wavy  threads,  1-1.5  /"  wide,  which 
branch  and  show  a  radial  growth,  as  in  actinomyces.  Club-shaped 
ends,  however,  are  absent  or  very  feebly  developed.  Fragmentation 
is  frequently  observed. 

ANILIN  DYES. — Stain  readily;   so  does  Gram's  method. 

GROWTH. — Is  rather  slow,  requiring  several  days  to  form  appre- 
ciable colonies.  It  grows  best  on  unneutralized  potato,  or  in  hay  in- 
fusions. 

Stab  culture. — In  gelatin  it  shows  a  slight  white  growth  along  the  line  of  inoculation 
and  on  the  surface. 

Streak  culture— -On  ordinary  agar  the  growth  is  feeble.  On  glucose  glycerin  agar  it 
forms  splendid,  raised,  rounded,  smooth  colonies  which  are  slightly  yellowish,  and  later 
develop  frequently  a  rose  or  bright  red  color.  These  colonies  adhere  to  the  surface  and  are 
very  hard,  as  in  the  case  of  actinomyces. 

Milk. — Is  slowly  peptonized,  but  is  not  coagulated. 

Potato. — It  forms,  on  about  the  5th  day,  small  whitish  colonies  which  slowly  de- 
velop, forming  a  nodular  growth.  The  center  of  each  nodule  is  depressed.  In  old  tubes, 
especially  on  acid  potatoes,  a  pink  or  deep  red  color  may  develop.  A  similar  color  develops 
when  grown  on  carrots. 

Bouillon. — After  a  lapse  of  2  or  3  weeks  small,  whitish  balls  form  on  the  bottom. 
Vegetable  infusions  are  better  than  ordinary  bouillon.  The  growth  then  develops  in  4  or  5 
days,  as  small  floccules,  which  later  become  as  large  as  a  pea.  The  liquid  does  not  become 
cloudy.  May  grow  on  the  surface  of  bouillon  forming  a  growth  like  that  of  the  tubercle 
bacillus. 

RESISTANCE. — Vitality  unimpaired  after  drying  on  paper  for  9 
months,  or  being  kept  as  a  potato  culture  for  21  months.  The  ovoid 
spores  form  on  the  surface,  in  contact  with  air,  and  are  easily  colored 
with  simple  stains,  or  by  Gram's  method.  They  resist  heating  at  75° 
for  5  minutes. 

OXYGEN  REQUIREMENTS. — It  is  aerobic  and  does  not  grow  in  vacuo, 
in  COa  or  in  illuminating  gas. 

TEMPERATURE. — The  optimum  is  at  37°,  but  it  may  grow  at  ordin- 
ary temperature. 

BEHAVIOR  TO  GELATIN.— It  does  not  liquefy. 

PATHOGENESIS. — Pure  cultures  of  the  original  grains  injected  sub- 
cutaneously  or  intravenously,  or  into  the  peritoneal  cavity  are  with- 
out effect  in  the  guinea-pig,  rabbit,  dog,  sheep,  pigeon  and  chicken. 

The  above  organism  was  isolated  from  the  yellowish  caseous 
granules  mentioned  above.  Recently,  Wright  has  succeeded  in  grow- 
ing a  streptothrix  from  the  black  granules.  In  this  case  the  hyphae 
were  3-8  n  in  diameter.  Fragmentation  was  present,  and  in  old 
cultures  black  granules  or  sclerotia  developed.  It  was  likewise  non- 
pathogenic  to  animals. 

418 


DRAWINGS.  419 


Streptothrix  Farcinica,  Nocard  (1888). 


ORIGIN. — It  is  present  in  the  pus  from  the  subcutaneous  nodules, 
also  in  the  pseudo-tubercles  in  the  lung's  and  other  internal  organs  in 
the  disease  known  as  cattle-farcy  (farcin  du  boeuf).  The  disease  is  very 
rare  in  Prance;  apparently  quite  common  on  the  Island  of  Guadeloupe. 

FORM. — The  organism  in  diseased  tissue  and  in  pure  culture  forms 
slender  filaments  which  in  width  are  comparable  to  the  rouget  bacil- 
lus. The  filaments  are  densely  interwoven,  forming-  masses.  They 
branch  dichotomously. 

ANILIN  DYES. — It  does  not  stain  like  the  tubercle  bacillus.  In 
Gram's  method,  if  treatment  with  alcohol  is  continued  it  decolors.  It 
is  stained  by'the  Gram-Weigert  method  (decoloration  in  anilin  oil). 

GROWTH. — Develops  readily  on  solid  and  liquid  media  at  the  body 
temperature.  Acid  media  are  not  favorable.  Cultures  kept  at  40° 
retain  their  vitality  for  months.  No  growth  in  the  absence  of  air. 

Streak  culture.— On  agar  it  forms  irregular,  raised,  yellowish  masses  which  unite 
and  form  a  thick,  coarsely  folded  membrane,  in  appearance  not  unlike  that  produced  by 
the  tubercle  bacillus. 

On  potato,  it  forms  raised,  dry,  yellowish  scales  and  the  growth  as  a  whole  resembles 
that  of  the  tubercle  bacillus.  On  blood-serum,  the  growth  is  slow  and  has  the  same  charac- 
teristics as  that  on  potato. 

Bouillon. — Cultures  show  irregular  masses  which  usually  fall  to  the  bottom.  When, 
however,  the  growth  develops  on  the  surface  it  presents  the  same  characteristics  as  the 
tubercle  bacillus.  It  does  not  coagulate  milk  or  alter  its  reaction. 

RESISTANCE. — Old  growths  on  the  surface  of  glycerin  bouillon  are 
said  to  contain  extremely  small  ovoid  spores  which  resist  staining . 
These  may,  however,  be  the  result  of  fragmentation.  10  minutes 
heating  at  70°  destroys  the  organism. 

TEMPERATURE. — Does  not  grow  at  ordinary  temperature,  but  does 
grow  at  30  to  40°. 

OXYGEN  REQUIREMENTS. — Is  aerobic;  will  not  grow  in  vacuo  or 
in  CO2. 

PATHOGENESIS. — The  guinea-pig  is  the  most  susceptible  animal, 
then  come  cattle  and  sheep.  The  rabbit,  dog,  cat  and  horse  seem  to 
be  refractory.  Intravenous  and  intraperitoneal  injections  produce  in 
from  9  to  20  days  miliary  pseudo-tubercles  which  cover  the  serous  sur- 
faces, and,  in  the  former  case,  they  are  also  present  in  the  various 
organs.  When  inoculated  subcutaneously  in  guinea-pigs  or  other 
susceptible  animals,  a  local  abscess  forms,  which  discharges  and 
secondary  ones  form  slowly.  This  infection,  like  the  natural  one, 
rarely  produces  death. 

420 


DRAWINGS.  421 


CHAPTER  XIII. 
EXAMINATION  OF  WATER,  SOIL  AND  AIR. 

A  pure  water-supply  is  of  first  importance  in  the  con- 
servation of  public  health.  That  impure  water  is  the  cause 
of  many  diseases  is,  to-day  a  well  recognized  fact.  It  has 
ceased  to  be  a  theory.  Cholera  and  typhoid  fever  are 
striking  examples  of  water-borne  diseases.  It  should  not 
be  inferred,  however,  that  these  diseases  are  always  con- 
veyed through  drinking-water.  The  specific  germs  may,  at 
times,  be  introduced  into  the  body  by  means  of  various 
articles  of  food  or  by  direct  contact  of  the  mouth  with 
infected  articles.  Nevertheless,  the  most  common  vehicle  by 
which  these  organisms  enter  the  body  is  the  drinking-water. 

A  pure  water,  therefore,  is  essential  to  the  prevention 
of  these  and  other  diseases.  Until  very  recent  times  an 
opinion  as  to  the  purity  of  a  water  was  based  wholly  upon  a 
chemical  examination.  In  some  instances,  the  water  may 
be  injurious  because  of  the  presence  of  poisonous  metals 
or  other  injurious  compounds.  Thus,  the  presence  of 
arsenic  or  lead,  or  of  large  amounts  of  copper  or  zinc  would 
certainly  render  the  water  impure  and  highly  dangerous  to 
health.  In  such  cases  the  chemical  examination  will  di- 
rectly reveal  the  presence  of  the  poisonous  metal.  As  a 
striking  illustration  of  this,  may  be  mentioned  the  fact  that 
Vaughan  detected  relatively  large  amounts  of  arsenic  and 
antimony  in  the  river  water  of  a  Western  mining  region. 
The  water  had  been  used  by  animals,  a  large  number  of 
which,  as  a  result,  died  from  arsenical  poisoning. 

The  real  danger  in  water,  however,  does  not  lie  in  the 
presence  of  poisonous  chemical  substances.  Intoxications, 


EXAMINATION  OF   WATER.  423 

such  as  mentioned,  are  extremely  rare.  On  the  other  hand, 
infections  through  impure  water  are  unfortunately  but  too 
common.  By  infection  is  meant  the  introduction  into  the 
body  of  a  specific,  pathogenic,  micro-organism.  When  im- 
pure water  is  spoken  of,  it  does  not  follow  that  the  water 
is  unsightly,  malodorous  or  repulsive  to  the  taste.  The 
water  may  be  perfectly  clear,  sparkling  in  character  and  a 
chemical  examination  may  show  that  it  contains  minimal 
amounts  of  organic  and  inorganic  constituents,  and  yet,  that 
water  may  be  impure  because  of  the  presence  of  pathogenic 
bacteria. 

A  chemical  examination  will  not  detect  the  presence  of 
bacteria,  much  less  assist  in  their  identification.  It  can, 
however,  detect  the  presence  of  certain  chemical  substances 
from  the  relative  amounts  of  which  an  inference  may  be 
drawn  as  to  the  existence  of  pollution  with  human  or 
animal  excreta.  A  pollution  of  this  kind  once  established 
indicates  that  danger  from  infection  may  be  expected  when- 
ever the  pathogenic  germ  is  actually  present  in  such  excreta. 
From  a  chemical  standpoint  a  good  water  should  not  con- 
tain more  of  the  several  constituents,  in  parts  per  million 
or  mg.  per  liter,  than  what  is  shown  in  the  following  table: 

Total  residue 500  Nitric  anhydride 5-15 

Earthy  bases *. . .  180-200  Nitrous  acid traces 

Sulphuric  anhydride ..  80-100  Albuminoid  ammonia.      0.20 

Chlorine 20-30 

Moreover,  it  should  not  consume  more  than  8  to  10  parts, 
per  liter,  of  potassium  permanganate. 

It  should  not  be  understood  from  these  requirements 
that  a  water  containing  an  excess  of  these  constituents 
above  the  limits  given  will  necessarily  be  dangerous.  The 
chlorine,  nitrates,  nitrites  and  ammonia  are  in  themselves- 
harmless  and  can  be  taken  with  impurity  in  relatively 
large  doses.  The  data  given,  however,  are  supposed  to 
represent  the  average  composition  of  good  water.  An 


424  BACTERIOLOGY. 

excess  of  chlorine  and  nitrogenous  matter  would  therefore 
indicate  the  presence  of  animal  excreta.  An  increase,  for 
instance,  in  the  amount  of  chlorine  might  be  due  to  the 
presence  of  sodium  chloride  derived  from  urine.  Similarly, 
large  quantities  of  free  and  albuminoid  ammonia  are 
usually  taken  to  indicate  the  presence  of  urea.  Unchanged 
urea  would  be  represented  by  albuminoid  ammonia.  But, 
as  is  known,  urea  readily  undergoes  ammoniacal  fermenta- 
tion and  yields  ammonia  and  carbonic  acid.  Other  bacteria 
attack  the  ammonia  and  convert  the  nitrogen  present  into 
nitrous  and  nitric  acids.  For  these  reasons  an  increase  in 
free  and  albuminoid  ammonia,  and  in  nitrates  and  nitrites 
is  taken  as  indicating  a  pollution  with  organic,  nitrogenous, 
waste  matter. 

The  assumption  of  the  existence  of  a  pollution  is  not 
always  justifiable  whenever  the  limits,  as  given  above,  are 
exceeded.  Thus,  in  Michigan  it  is  not  uncommon,  on  ac- 
count of  the  peculiar  geological  formation,  to  meet  with  a 
marked  increase  in  the  amount  of  chlorine  or  salt.  More- 
over, in  many,  especially  low  lying  sections,  the  water  will 
be  rich  in  nitrogenous  constituents  which  in  this  case  are 
derived  from  vegetable  and  not  animal  matter. 

It  is  evident  from  what  has  been  said  that  a  chemical 
analysis  may,  or  may  not,  indicate  the  presence  of  pollution 
with  animal  excreta.  Furthermore,  the  important  fact 
should  not  be  overlooked  that  a  specific  organism  may  be 
present,  and  hence  render  a  water  extremely  dangerous 
without  any  appreciable  increase  in  the  chemical  consti- 
tuents of  such  water.  A  chemical  analysis  of  water  is  use- 
ful and  it  should  always  be  carried  out,  but  the  results  ob- 
tained should,  as  a  rule,  be  subordinated  to  those  obtained 
by  a  bacteriological  examination. 

A  bacteriological  examination  of  water  is  intended  to 
reveal  the  number  and  kind  of  bacteria  that  may  be  pres- 
ent. As  a  rule,  the  smaller  the  number  of  bacteria  in  a 


EXAMINATION  OF    WATER.  425 

given  volume,  the  more  likely  is  it  .to  be  free  from  in- 
jurious forms.  On  the  other  hand,  if  the  number  of  bac- 
teria is  subject  to  considerable  fluctuation  and  is  usually^ 
high,  it  indicates  conditions  favorable  to  decomposition. 
The  bacteria  in  a  given  specimen  of  water  may  be  ex- 
tremely numerous,  and  yet  they  may  be  mere  harmless 
saprophytes.  The  fact  that  such  water  is  a  good  nutrient 
medium  should  make  it  suspicious  inasmuch  as  intestinal 
bacteria,  if  once  introduced,  will  be  favored  in  like  manner. 
They  may  indeed  be  present  but,  masked  by  the  large 
number  of  common  water  bacteria,  they  may  escape  detec- 
tion. 

The  chief  interest  is  attached  to  the  kind  of  bacteria 
present.  The  recognition  of  certain  well  known  intestinal 
bacteria  in  a  given  water  may  be  taken  as  a  good  indica- 
tion of  the  existence  of  pollution  by  animal  excreta.  Thus, 
the  Bacillus  coli  communis  is  invariably  present  in  the  in- 
testinal contents  of  man  and  animals,  and  hence  its  detec- 
tion in  water,  especially  in  appreciable  numbers,  points  to 
the  source  of  the  contamination.  The  fact,  however, 
should  not  be  overlooked  that  this  or  related  species  are 
widely  distributed  in  nature.  It  makes  its  appearance  in 
the  intestines  of  the  new-born  within  a  few  hours  after 
birth  and  this  fact  in  itself  indicates  the  wide  prevalence 
of  the  colon  bacillus.  Consequently,  the  detection  of  the 
colon  bacillus  in  a  water  should  arouse  suspicion  and  cause 
a  thorough  examination  of  the  premises  with  reference  to 
the  close  presence  of  polluting  material.  The  finding  of 
the  colon  bacillus,  therefore,  does  not  indicate  more  than 
the  possibility  of  the  existence  of  pollution,  which,  as  a  rule, 
will  also  be  indicated  by  a  chemical  examination.  3D 

The  recognition  of  a  specific  pathogenic  germ  in  a 
water  is  of  prime  importance  inasmuch  as  it  demonstrates 
actual  pollution  as  well  as  the  possibility  of  infection.  The 
typhoid  bacillus  is  the  one  most  frequently  looked  for. 
Under  exceptional  conditions  the  cholera,  anthrax  and 


426  BACTERIOLOGY. 

pus -producing1  germs  are  included  in  the  scope  of  a  water 
examination. 

Typhoid  bacillus. — The  typhoid  bacillus  when  once  intro- 
duced into  a  water-supply  does  not  necessarily  remain  in 
such  water  for  a  considerable  length  of  time.  The  results 
of -many  experiments  made  by  different  observers  would  in- 
dicate that  the  typhoid  bacillus  when  introduced  into  or- 
dinary water  may  remain  alive  for  about  a  week.  This  is 
about  the  limit  when  they  are  placed  in  very  pure  water  kept 
at  a  low  temperature,  i.  e. ,  under  conditions  very  unfavor- 
able to  the  vitality  of  the  org-anism.  This  applies  to  natural 
as  well  as  distilled  water,  whether  sterilized  or  unsterilized. 

When  the  temperature  is  above  10°,  and  especially,  when 
the  water  contains  an  appreciable  quantity  of  organic  mat- 
ter, the  typhoid  bacilli  may  persist  for  a  much  longer  per- 
iod. The  organisms  under  these  conditions  may  actually 
increase  in  number  and  retain  their  vitality  for  weeks  and 
months.  Hence  an  impure  water,  as  indicated  by  a  chem- 
ical analysis,  is  to  be  regarded  as  suspicious  because  it  may 
favor  the  growth  and  persistence  of  typhoid  germs  when 
once  introduced. 

The  recognition  of  the  typhoid  bacillus  in  water  is  by 
no  means  an  easy  matter.  Usually  an  examination  is  called 
for  after  an  outbreak  of  the  disease  has  taken  place.  That 
is  to  say,  the  period  of  incubation,  which  extends  from  1  to 
2  weeks,  is  allowed  to  pass  before  a  search  is  instituted  for 
the  organism.  This  time  is  sufficient,  under  conditions  in- 
dicated above,  for  the  typhoid  germ  to  disappear,  and,  con- 
sequently, the  examination  in  such  cases  will  be  negative. 
Moreover,  even  in  water  which  in  the  beginning  was 
favorable  to  the  growth  of  this  organism,  the  detection  is 
still  difficult.  At  the  time  of  the  examination,  the  germs 
may  already  be  dying  out  and  decreasing  in  number.  The 
small  volume  of  water  which  is  necessarily  taken  for  analy- 
sis may,  or  may  not,  be  a  perfect  sample  of  the  whole. 


EXAMINATION   OF    WATER.  427 

Chemically  speaking*,  it  is  a  good  sample  because  the  solu- 
ble constituents  in  one  drop  are  present  in  relatively 
the  same  amounts  as  in  a  liter  or  barrel  of  the  water.. 
This  is  not  true  with  reference  to  the  suspended  bacteria. 
These  are  solid  particles,  and  as  such  tend  to  settle  or 
subside.  The  so-called  water  purification  in  many  instances 
may  largely  consist  of  mechanical  sedimentation.  Al- 
though, therefore,  the  mass  of  the  water  may  still  contain 
some  typhoid  bacilli,  the  small  quantity  taken  for  examin- 
ation (1-10-20  drops),  may,  or  may  not,  contain  one  or  more 
of  these  organisms.  The  few  specific  germs  are  easily 
masked  by  the  hundreds  and  thousands  of  common  water 
bacteria  that  are  usually  present.  If  necessary,  the  num- 
ber of  organisms  taken  for  an  examination  may  be  increased 
by  prolonged  centrifugation  of  the  water.  Moreover,  the 
separation  may  be  favored  by  the  addition  of  typhoid  serum 
or  of  histon. 

The  typhoid-like  or  pseudo-typhoid  bacilli  that  may  be 
present  require  careful  and  prolonged  study.  Recent  inves- 
tigations have  shown  that  typhoid  bacilli,  or  at  all  events 
organisms  that  cannot  be  distinguished  from  these  by  any 
known  test,  may  be  present  in  water,  in  soil;  also  in  the  feces 
of  healthy  persons  who  have  never  had  typhoid  fever. 
Thus,  Losener  isolated  5  such  organisms  from  tap-water, 
field  soil,  normal  pus,  from  a  hog  cadaver  buried  4  weeks, 
and  from  a  typhoid  spleen  buried  96  days.  Pfeiffer  and 
Kolle  have  pronounced  these  organisms  to  correspond  ex- 
actly to  the  typhoid  bacillus,  even  to  the  application  of 
Pfeiffer's  reaction,  and  hence  their  typhoid  character  cannot 
be  questioned.  Remlinger  and  Schneider,  employing  Eis- 
ner's medium  and  working  under  the  direction  of  Vaillard 
at  the  Val-de-Grace  hospital  at  Paris,  isolated  a  bacillus  in- 
distinguishable from  that  of  Eberth: 

8  times  out  of  36  samples  of  water; 

6  times  out  of  10  samples  of  soil; 

3  times  out  of  8  samples  of  normal  feces. 


428 


BACTERIOLOGY. 


Of  the  18  pseudo- typhoid  bacilli  isolated,  12  killed  guinea 
pigs  when  injected  intraperitoneally  in  doses  of  2  c.c., 
whereas  the  remaining  6  had  no  effect.  Anti-typhoid  serum 
was  said  to  be  efficacious  in  preventing  infection  by  these 
organisms.  A  mention  may  be  made,  moreover,  of  the  fact 
that  Ohlmacher  isolated  a  bacillus,  indistinguishable  from 
that  of  Eberth,  from  the  Cleveland  tap-water.  In  cadavers 
buried  for  1^-2  years  only  typhoid-like  bacteria  are  said  to 
be  present. 

The  typhoid  bacillus  cannot  be  identified  by  any  one 
characteristic.  The  suspected  culture  must  give  most,  if 
not  all,  of  the  known  reactions  of  the  typhoid  germ.  These 
reactions  may  be  grouped  together  in  the  following  sum- 
marv: 


1. — Appearance  of  colonies. 

2. — Active  motion. 

3. — Large  number  of  whips,  in- 
cluding- giant- whips. 

4.— Gram's  stain  negative. 

•5.— No  gas  production. 

•6. — No  coagulation  of  milk. 

7. — No  indol  reaction. 

8. — No  acid  production  (lactose 
media). 


9. — Invisible  growth  on  potato. 

10. — Colonies  on  Eisner's  medium. 

11. — Transparent  diffusion  on  Stod- 
dart's  medium. 

12. — No  growth  in  Uschinsky's  fluid. 

13.— Agglutination  by  diluted 
typhoid  fever  serum. 

14.— Pfeiffer's  reaction  with  anti- 
infectious  typhoid  serum. 

15.— Pathogenic  effects. 


The  above  characteristics  will  be  found  described  un- 
der their  respective  heads.  The  ubiquitous  colon  -  bacillus 
is  especially  ruled  out  by  the  last  11  tests.  The  colon  ba- 
cillus is  less  pathogenic  and  rarely  kills  guinea-pigs  when 
injected  subcutaneously. 

Plate  cultures  can  be  made  with  ordinary  gelatin,  with 
Eisner's  medium  or  with  a  3-5  per  cent,  urine  gelatin  (see 
Chapter  XIV).  The  suspected  typhoid  colonies  are  then 
transplanted  to  other  media  and  subjected  to  the  tests 
given  above.  It  is  advisable  to  transplant  20  or  more  sus- 
picious looking  colonies  into  bouillon  tubes  which  are  then 
placed  at  39°  to  develop.  A  drop  or  two  of  each  culture 


EXAMINATION   OF    WATER.  429 

that  now  develops  is  placed,  by  means  of  a  drawn-out  tube 
pipette  (Fig.  61),  into  sterile  milk  tubes.  The  inoculated 
milk  tubes  are  then  placed  at  39°  for  1-3  days.  As  a 
trol  of  the  sterility  of  the  milk  a  number  of  the  uninocula- 
ted  tubes  should  likewise  be  placed  in  the  incubator. 
Moreover,  it  is  advisable  to  inoculate  a  like  number  of 
milk  tubes  with  a  known  typhoid  bacillus  in  order  to  elim- 
inate a  possible  error  (p.  163),  which  might  arise  if  spores 
of  anaerobes  were  present.  The  milk  tubes  that  coagu- 
late can  be  discarded  at  once,  together  with  ihe  bouillon 
cultures  from  which  they  were  inoculated.  The  milk  tubes 
that  do  not  coagulate  may,  or  may  not,  contain  the  Eberth 
bacillus.  The  bouillon  cultures  from  which  these  tubes 
were  inoculated  can  now  be  used  for  the  supplementary 
tests. 

The  procedure  as  outlined  is  preferable  to  direct  inocu- 
lation into  milk  fr*om  the  suspected  colonies.  The  trans- 
plantation in  the  latter  case  may  sometimes  fail  to  de- 
velop or  does  so  very  slowly.  When  bouillon  cultures  are 
grown,  the  inoculations  can  be  made  with  liberal  amounts  of 
the  organism  and  the  original  material  is  saved  for  subse- 
quent tests. 

At  times  it  may  be  more  advantageous  to  make  a  sur- 
face touch  colony  on  Stoddart's  medium  contained  in  a 
large  flask  or  Esmarch  dish.  The  transparent  border 
growth  can  then  be  plated  on  gelatin  or  on  Eisner's  me- 
dium, and  the  suspicious  colonies  can  be  tested  as  in 
the  manner  indicated  above.  The  addition  of  a  drop  of  a 
bouillon  culture  to  a  liter  of  tap-water  can  be  detected 
quite  readily  in  this  way.  The  methods  mentioned  above 
are  also  resorted  to  when  isolating  the  Eberth  bacillus 
from  the  urine  or  f eces  of  typhoid  patients. 

Cholera  vibrio. — This  organism  has  been  repeatedly 
found  in  water  during  times  of  cholera  epidemics.  As  in 
the  case  of  the  Eberth  bacillus,  the  detection  of  the  comma 


430  BACTERIOLOGY. 

bacillus  is  not  an  easy  matter.  The  cultural  and  morpho- 
logical properties  are  subject  to  considerable  variation. 
Moreover,  closely  similar  organisms  may  exist  in  river 
water.  These  water  vibrios  phosphoresce,  but  this  prop- 
erty as  in  the  case  of  other  photogenic  bacteria  may  disap- 
pear on  cultivation.  It  is  doubtful  whether  the  true  cholera 
vibrio  can  phosphoresce. 

The  recognition  of  the  cholera  vibrio  in  water  or  in 
suspected  discharges  is  based  upon  the  following  charac- 
teristics: 

1. — Microscopical  appearance.         5. — Agar  and  gelatin  tube  cul- 

2. — Cultures  in  Dunham's  solution.  tures. 

3. — Appearance  of  colonies  on        6.  — Positive  indol  reaction. 

gelatin  plates.  7. — Inoculation  of  guinea-pigs. 

4. — Ap'pearance  of  colonies  on        8. — Pfeiffer's  reaction. 

agar  plates.  9.     Agglutination. 

In  the  case  of  suspected  intestinal  contents,  the  micro- 
scopical examination  may  in  a  few  minutes  justify  a  diag- 
nosis of  cholera,  but  a  positive  diagnosis  can  only  be  ob- 
tained by  confirming  all  the  characteristics  of  the  organ- 
ism. Under  favorable  conditions  this  may  be  accomplished 
in  less  than  24  hours.  A  drop  of  the  intestinal  liquid  should 
be  spread  over  the  cover-glass,  or  better  still,  one  of  the 
numerous  flakes  present  in  the  rice-water  discharge  should 
be  thoroughly  rubbed  over  the  cover-glass.  By  staining 
with  carbolic  fuchs(in,  the  presence  of  comma-shaped  or- 
ganisms can  be  readily  established  if  they  are  present  in 
large  numbers. 

It  should  be  remembered,  however,  that  the  cholera  vib- 
rio may  disappear  from  the  intestines  after  about  the  fifth 
day.  In  other  instances,  only  a  few  cholera  vibrios  may  be 
present  and  these  may  escape  detection,  owing  to  the  large 
number  of  other  organisms  that  occur  in  the  material. 

Even  when  only  a  few  cholera  germs  are  present  in  the 
•discharge  or  in  the  water,  they  can  be  brought  to  light  by 
a  peculiar  method  of  "accumulation."  Thus,  when  planted  in 


EXAMINATION   OF   WATER.  431 

Dunham's  solution  the  vibrio  grows  rapidly,  and  on  account 
of  its  extreme  aerobic  tendency  it  accumulates  or  gathers 
on  the  surface  of  the  liquid  forming-  a  broken  pellicle.  Dun- 
ham's solution  is  water  to  which  1  per  cent,  pepton  and  0.5 
per  cent,  salt  has  been  added  (p.  344).  A  modification  by 
Metchnikoff  contains  1  per  cent,  each  of  pepton  and  salt, 
and  2  per  cent,  of  gelatin. 

About  100  c.c.  of  the  liquid  are  placed  in  each  of  several 
Erlenmeyer  flasks.  These  are  then  inoculated  and  set  aside 
at  37°.  In  from  10-20  hours  the  surface  growth  is  examined. 
A  loopful  can  be  used  for  staining-  and  for  hanging-drop  ex- 
amination. At  the  same  time  gelatin  and  agar  plates 
should  be  made  from  this  surface  pellicle. 

The  gelatin  plates  should  be  developed  at  as  high  a 
temperature  (22-24°)  as  possible,  without  melting  the  gela- 
tin. This  can  be  done  very  satisfactorily  by  keeping  the 
plates  in  the  water-cooler  (Pig.  33,  p.  179).  The  agar  plates 
are  not  prepared  in  the  ordinary  way.  Koch's  procedure 
consists  of  pouring  the  agar  into  Petri  dishes  where  it  is 
allowed  to  solidify  and  remain  for  several  days.  This  is  to 
allow  the  water  of  condensation  to  evaporate.  The  surface 
of  the  agar  plates,  thus  prepared,  is  repeatedly  streaked 
with  a  platinum  wire.  The  organisms  are  thus  planted  on 
the  surface  and  hence  good  isolated  colonies  can  be  obtained. 
The  agar  plates  are  placed  at  37°  and  in  urgent  cases  they 
can  be  examined  in  about  8  or  10  hours. 

The  characteristic  colonies  on  gelatin  or  on  agar  are 
transplanted  to  Dunham's  solution  in  tubes^  and  to  agar  and 
gelatin  tubes.  The  first  two  media  are  then  placed  at  37°. 
In  about  10  hours  the  indol  reaction  may  be  applied  to  the 
culture  in  Dunham's  solution.  A  loopful  of  the  growth 
(2  mg.)  on  inclined  agar  can  be  removed  at  the  end  of  about 
20  hours,  suspended  in  1  c.c.  of  bouillon  and  this  then  injec- 
ted into  the  peritoneal  cavity  of  a  guinea-pig.  The  tem- 
perature and  the  weight  of  the  animal  should  be  taken  be- 
fore the  injection.  The  former  should  again  be  taken  every 


432  BACTERIOLOGY. 

2  hours  after  the  injection.  In  a  few  hours  the  animal  be- 
comes sick,  the  temperature  drops  gradually  to  30°  and 
death  eventually  results. 

In  the  case  of  water  which  contains  but  a  very  few  cholera  vib- 
rios, instead  of  adding  a  small  amount  to  the  Dunham's  solution,  it 
is  better  to  add  the  necessary  amount  of  pepton  and  salt  to  300  c.c.  or 
more  of  the  water,  thus  converting  the  suspected  water  into  Dunham's 
solution.  These  constituents  can  best  be  introduced  by  adding  the 
calculated  amount  of  a  sterile  solution  containing  20  per  cent  of  pep- 
ton  and  10  per  cent,  of  NaCl,  i.  e.,  5  c.c.  per  100  c.c.  of  water.  It 
should  then  be  placed  in  a  large  flask,  so  as  to  have  as  large  a  surface 
as  possible,  and  allowed  to  develop  at  37°.  The  subsequent  examina- 
tions are  the  same  as  those  outlined  above. 

It  must  not  be  expected  that  the  cholera  vibrio  which 
has  been  isolated  will  agree  in  every  respect  with  the  class- 
ical description  of  this  organism.  On  the  contrary,  varieties 
must  be  expected  inasmuch  as  pleomorphism  is  more  marked 
in  the  case  of  the  cholera  germ  than  in  any  other  known 
species.  Usually  it  is  a  short,  thick,  bent  rod,  but  it  is  pos- 
sible to  have  long-,  slender,  almost  straight  varieties.  Usu- 
ally it  possesses  but  one  whip,  but  some  have  been  shown 
to  possess  as  many  as  four  flagella.  Moreover,  although  it  is 
usually  exceedingly  motile,  varieties  may  be  found  that  are 
motionless.  Some  varieties  will  coagulate  milk,  others  will 
not.  Again,  as  a  rule,  the  liquefaction  of  gelatin  is  slow, 
whereas  some  liquefy  this  medium  very  rapidly.  The  indol 
reaction  and  virulence,  which  are  especially  relied  upon  in 
an  identification,'  are  likewise  subject  to  extreme  variation. 
Pfeiffer's  phenomenon  is  described  in  Chapter  XIV.  It 
affords  as  good  a  means  of  differentiation  as  any  known 
procedure. 

The  cholera  vibrio  may  retain  its  vitality 'in  water  for 
a  considerable  period.  It  has  been  kept  in  sterile  tap-water 
for  more  than  a  year.  It  dies  out  rapidly  in  water  which  is 
kept  at  or  near  the  freezing-point.  Certain  varieties,  how- 
ever, can  resist  actual  freezing  for  many  days.  As  a  rule,. 


EXAMINATION   OF    WATER.  433 

they  will  live  much  longer  in  water  having  the  ordinary 
room  temperature  of  about  20°.  The  interesting"  studies  of 
Hankin  have  shown  that  the  water  of  certain  rivers  in  In- 
dia will  destroy  the  cholera  germ  in  3  hours;  whereas,  if  the 
water  is  previously  boiled  it  will  have  no  such  effect.  Simi- 
lar germicidal  substances  may  be  present  at  times  in  the 
water  of  other  localities  and  thus  explain,  at  least  in  part, 
the  so-called  local  immunity. 


Water  Analysis. 

A  bacteriological  analysis  of  .water  consists:  (1)  in  the 
determination  of  the  number  of  bacteria;  (2)  the  identifica- 
tion of  the  several  species;  (3)  the  recognition  of  the  patho- 
genic bacteria  present. 
i 

Number  of  bacteria. — The  water  to  be  examined  should 
be  received  in  a  sterilized  bottle  or  flask  and  thoroughly 
protected  against  subsequent  contamination.  Furthermore, 
in  view  of  the  rapid  multiplication  of  bacteria,  a  given 
sample  of  water  should  be  examined  as  soon  as  possible 
after  collection.  The  method  commonly  employed  in  the  de- 
termination of  the  number  of  bacteria  present  is  as  follows: 

Several  1  c.c.  pipettes,  graduated  in  iV  c.c.,  are  placed 
in  a  pipette  box  and  sterilized  in  the  dry-heat  oven  in  the 
usual  way.  3  gelatin  tubes  are  then  liquefied  and  marked. 
By  means  of  a  sterilized,  cooled  pipette  1  e.c.  of  the  water 
is  transferred  into  tube  No.  1.  In  like  manner  %  c.c.  and  1 
drop  are  placed  into  tubes  2  and  3,  respectively.  The  con- 
tents of  the  tubes  are  gently  agitated  to  secure  complete 
mixture.  The  gelatin  is  then  poured  on  sterilized  glass 
plates  or  into  Petri  dishes,  observing  the  usual  precautions 
in  making  plate  cultures  (p.  175).  The  gelatin  plates  thus 
obtained  are  set  aside  for  two  or  three  days  at  18-20°  and 
and  the  colonies  which  develop  are  then  counted. 


434  BACTERIOLOGY. 

Inasmuch  as  the  bacteria  are  liable  to  multiply  rapidly,  especial- 
ly if  the  water  is  taken  from  a  cool  source  and  is  then  kept  at 
ordinary  temperature,  it  is  advisable  to  plate  the  water  at  the  time 
it  is  collected.  Under  these  circumstances,  instead  of  plates  or  Petri 
dishes,  flat  flasks  or  bottles  can  be  used..  These  contain  the  requisite 
amount  of  sterile  gelatin  which  is  inoculated  with  the  water  as  soon 
as  it  is  drawn.  The  flask  is  placed  on  its  side,  and,  when  the  gelatin 
solidifies,  it  can  be  taken  back  to  the  laboratory. 

If  only  a  small  number  of  colonies  are  present  they 
can  be  counted  with  the  unaided  eye,  but  when,  as  it  fre- 
quently happens,  the  number  is  very  large,  it  is  desirable 
to  make  use  of  a  counting  apparatus.  Fig".  57  shows  the 
counting"  apparatus  of  Wolffhugel,  which  is  usually  em- 
ployed when  ordinary  glass  plates  are  used.  The  gelatin 
plate,  on  which  the  colonies  are  to  be  counted,  is  placed  on 
the  black  glass  base  and  covered  with  a  glass  plate  ruled 
into  squares.  The  number  of  colonies  under  each  square 
can  thus  be  easily  determined.  When  possible,  the  number 
of  colonies  under  each  square  should  actually  be  counted. 
As  a  rule,  however,  it  is  customary  to  count  the  number  of 
colonies  found  under  each  of  6,  8  or  10  squares  selected  at 
random  from  over  the  surface  of  the  plate.  The  average 
number  present  in  one  square  is  then  ascertained. 


FIG.  57.     Wolffhiigel's  apparatus  for  counting  colonies. 

The  total  number  of  colonies  on  the  plate  is  found  by 
determining  the  number  of  square  centimeters  which  the  gel- 
atin on  the  plate  covers,  and  multiplying  this  figure  by  the 
average  number  of  colonies  per  square.  Since  each  colony 


EXAMINATION    OF    WATER.  435 

is  derived  from  a  single  cell  this  figure  then  represents  the 
number  of.  bacteria  present  on  the  plate  examined.  If  this 
plate  is  made  from  the  tube  to  which  1  c.c.  of  water  was 
added  then  the  results  are  expressed,  at  once,  as  so  many 
bacteria  per  c.c. 

In  case  the  plate  is  made  with  the  gelatin  that  re- 
ceived £  c.c.  of  water  the  result  is  multiplied  by  2.  In 
order  to  express  the  results  obtained  with  the  plate  con- 
taining 1  drop  of  water,  it  is  necessary  to  know  how  many 
drops  the  particular  pipette  employed  will  discharge  from 
1  c.c.  of  water.  Drops  vary  a  great  deal  in  size  but,  on  an 
average,  1  c.c.  will  yield  20  drops.  As  indicated  above,  the 
results  should  always  be  expressed  as  so  many  bacteria 
per  c.c. 

Wolffhiigel's  apparatus  can  also  be  employed  to  count  the  number 
of  colonies  in  a  Petri  dish.  The  latt.er,  if  possible,  should  be  inverted 
and  the  ruled  plate  then  brought  into  contact  with  the  bottom  of 
the  dish.  The  average  number  of  colonies  per  square  are  determined 
as  above.  Since  each  square  is  actually  1  cm.  square,  it  is  necessary 
now  to  determine  the  number  of  square  centimeters  covered  by  the 
gelatin  in  the  dish.  The  area  of  a  circle  is  TT  R?.  Hence,  on  multi- 
plying the  square  of  the  radius  by  3.1416  we  obtain  the  area  of  the 
circle  expressed  in  square  cm.  This,  multiplied  by  the  average  number 
of  colonies  per  square,  will  give  the  number  of  colonies  on  the  plate. 

Several  modifications  of  the  above  apparatus  have  been  devised 
especially  for  use  in  connection  with  Petri  dishes.  That  of  Laf ar 
(Fig.  58)  is  widely  used.  It  consists  of  5  concentric  rings  which  are 
divided  by  18  radii.  Each  of  the  several  spaces,  thus  resulting,  has 
an  area  of  1  sq.  cm.  Three  of  the  sectors  are  still  further  subdivided 
in  order  to  facilitate  the  counting  of  colonies  when  very  numerous. 
The  Petri  dish,  unlike  the  ordinary  gelatin  plate,  is  not  strictly  flat. 
The  center  is  almost  invariably  slightly  raised,  and,  as  a  result,  the 
medium  over  the  center  will  be  thinner  than  that  near  the  edge. 
Obviously,  the  number  of  colonies  in  a  square  cm.  over  the  center 
will  be  considerably  less  than  in  one  farther  removed  from  this  point. 
It  is,  therefore,  not  advisable  to  count  at  random  the  number  of 
colonies  in  a  square  cm.,  as  in  the  case  of  the  WoliThiigel  apparatus. 
The  counting  should  be  done  by  sectors.  The  lines  are  etched  on  a 
glass  plate  which  is  fixed  in  a  brass  collar.  The  bottom  of  the  Petri 


436 


BACTERIOLOGY. 


dish,  the  diameter  of  which  must  not  exceed  9.5  cm.,  is  placed  in  this 
collar  and  is  then  wedged,  so  as  to  be  immovable.  A  somewhat  simi- 
lar counter  (Park  or  Jeffer)  ruled  on  paper  can  be  obtained  at  very 
little  expense,  and  is  to  be  preferred  to  the  glass  plate  of  Laf ar. 


a  b 

FIG.  58.    a— Lafar's  counter;  b— Jeffers  modification. 

It  is  sometimes  desirable  to  have  an  approximate  idea  of  the 
number  of  colonies  on  a  Petri  dish,  when  their  number  is  so  great  as 
to  render  the  preceding-  method  impracticable.  In  such  cases  Buch- 
ner,  Neisser  and  others  determine  the  average  number  of  colonies 
present  in  the  field  of  a  microscope.  A  low  power  objective  (No.  3 
with  No.  1  ocular)  should  be  used  for  this  purpose.  The  diameter  of 
the  microscopic  field  must  be  known.  This  can  be  ascertained  by 
means  of  a  stage  micrometer  (p.  127)  or  in  a  manner  similar  to  that 
employed  in  the  measurement  of  objects  (p.  128).  The  field  is  pro- 
jected on  the  table  and  the  diameter  measured.  This  divided  by  the 
magnifying  power  employed  will  give  the  diameter  of  the  actual  field 
under  the  microscope.  Since  circles  are  to  each  other  as  the  squares 
of  their  radii,  the  area  of  the  large  circle  or  Petri  dish  can  be  easily 
obtained.  Thus,  if  the  radius  of  the  microscopic  field  is  .65  mm.;  that 
of  the  Petri  dish  46  mm.;  and  the  average  number  of  colonies  in  the 
microscopic  field  is  76;  then, 

r-  :      R2  :.  76  :  x 
.4225  :  2116  ::  76  :  x        x  =  380,000  colonies. 


EXAMINATION   OF    WATER.  437 

The  number  of  colonies  in  30  fields  should  be  counted  in  order  to 
obtain  a  good  average  number.  With  1,500  or  more  colonies  on  a 
plate  it  is  preferable  to  count  with  a  microscope.  This  is  espe- 
cially true  in  water  examinations  and  the  like  where  different  species 
are  present,  some  of  which  grow  rapidly  and  form  large  colonies 
whereas  others  grow  slowly  and  give  rise  to  very  small  ones.  .  A 
cross-wire  ocular  micrometer  should  be  used  when  the  number  of 
colonies  in  a  field  is  large.  The  number  of  colonies,  as  calculated  for 
the  whole  plate,  is  approximate  but,  if  the  work  is  properly  done,  the 
error  need  not  exceed  12  or  15  per  cent. 

In  case  the  number  of  bacteria  is  exceedingly  great,  the  Thoma- 
Zeiss  apparatus  for  counting  blood  corpuscles  can  be  used  to  deter- 
mine the  number  of  bacteria  present.  The  result  thus  obtained  will 
be  higher  than  that  obtained  by  plate  cultivation. 

To  obtain  accurate  results,  however,  it  would  be  necessary  to 
dilute  a  given  volume  of  the  water  with  a  known  volume  of  sterile 
water  and  then  plate  portions  of  this  mixture.  Thus,  1  c.c.  of  the 
water  might  be  added  to  99  c.c.  of  sterile  water,  and  portions  of 
1,  0.5,  0.25  and  0.1  c.c.  of  this  diluted  material  could  then  be  plated  in 
the  manner  described. 

The  number  of  colonies  counted  on  a  plate  are  taken  to 
represent  the  number  of  bacteria  present  in  the  water.  In 
reality,  this  represents  the  minimum  and  not  the  actual 
number  of  bacteria  present.  A  colony  may  be  derived  from 
several  cells.  Thus,  a  bacillus  growing-  in  pairs  or  in  short 
threads,  or  a  diplococcus  or  streptococcus  may  be  the  start- 
ing- point  of  the  colony.  Again,  the  conditions  of  cultiva- 
tion may  not  be  such  as  to  cause  the  development  of  all  the 
bacteria  that  may  be  present.  As  pointed  out,  heretofore, 
the  reaction  and  composition  of  the  gelatin,  as  well  as  the 
prevailing  temperature  will  influence  the  number  of  colon- 
ies that  develop.  Some  bacteria  may  be  present  that  will 
develop  only  at  the  temperature  of  the  body.  Moreover, 
the  method  takes  into  account  only  the  aerobic  bacteria. 
The  anaerobic  bacteria  present  will  not  develop.  Conse- 
quently, the  number  of  bacteria  present  in  a  water,  as  as- 
certained by  the  method  given,  is  merely  an  approximate 
number  and  serves  to  roughly  indicate  the  amount  of  or- 
ganic matter  present. 


438  BACTERIOLOGY. 

The  more  organic  matter  held  in  solution  in  a  water, 
the  more  suitable  it  is  as  a  culture  medium  for  bacteria. 
Such  a  water  will  not  only  permit  the  multiplication  of 
common  bacteria  but  will  also  favor  any  pathogenic  germ, 
like  the  typhoid  bacillus,  if  it  should  chance  to  be  intro- 
duced. The  same  interpretation  is .  to  be  given  to  a  large 
number  of  bacteria  in  a  water  as  is  given  to  a  large  amount 
of  organic  matter  determined  by  chemical  analysis. 

The  counting  of  bacteria  possesses  especial  value  in 
controlling,  from  time  to  time,  the  purity  of  a  water-sup- 
ply. A  large  and  persistent  increase  in  the  number  of 
bacteria  should  lead  to  an  investigation  of  the  cause. 
Where  the  water-supply  is  filtered,  the  daily  counting  of 
the  bacteria  in  the  filtered  water  will  give  the  best  indica- 
tions as  to  the  proper  working  of  the  plant. 

The  number  and  kind  of  species. — Apart  from  the  recogni- 
tion of  specific,  pathogenic  bacteria  very  little  need  be  said 
regarding  the  common  water  bacteria  present.  In  general, 
a  water  should  contain  but  a  few,  different  species.  When, 
for  instance,  10  or  more  different  species  are  present,  espe- 
cially if  each  is  represented  by  an  appreciable  number,  it 
would  serve  to  indicate  that  the  water  contains  consider- 
able organic  matter  and  is,  therefore,  a  good  medium 
for  the  growth  of  bacteria.  It  cannot  be  said  to  prove 
the  existence  of  pollution,  but  it  does  show  that  there 
are  conditions  favorable  to  bacterial  growth,  and  hence, 
favorable  to  pathogenic  bacteria  should  they  be  intro- 
duced.. 

Considerable  scientific  interest  is  attached  to  the  study 
of  the  different  species  of  water  bacteria,  but  apart  from 
this  they  require  no  attention  in  the  ordinary  routine  of 
water  analysis.  The  various  characteristics  of  a  given 
species  may  be  determined  by  making  a  microscopical  and 
cultural  study  of  the  organism  in  accordance  with  the 
methods  of  study  pursued  heretofore. 


EXAMINATION    OF    WATER.  439 

The  detection  of  the  typhoid  fever  and  cholera  bacilli 
has  been  discussed  in  the  preceding  pages  and  need,  there- 
fore, receive  no  further  attention  at  this  place.  In  addition 
to  these  organisms,  the  water  may  contain  bacteria  which 
are  highly  pathogenic  and  these,  consequently,  deserve  es- 
pecial consideration. 

Pathogenic  bacteria. — The  chief  object  of  the  bacterio- 
logical examination  of  water  is  to  determine  the  presence  or 
absence  of  pathogenic  or  toxicogenic  bacteria.  In  the 
method  as  ordinarily  carried  out,  this  is  done  by  recogniz- 
ing the  colony  of  the  specific  organism  sought  for.  When 
the  pathogenic  bacteria,  as  the  cholera  or  typhoid  fever 
bacillus  for  example,  are  present  in  large  numbers,  and  this 
is  very  rarely  the  case,  the  identification  can  perhaps  be 
easily  done.  On  the  other  hand  a  few  pathogenic  bacteria 
in  the  presence  of  a  large  number  of  saprophytic  organisms 
can  be  easily  overlooked,  and  in  such  cases  their  recogni- 
tion becomes  well-nigh  impossible.  In  view  of  these  facts 
the  following  method  was  devised  by  Vaughan,  and  has 
been  used  in  this  laboratory  since  1888.  It  is  based  upon 
the  fact  that  a  large  number  of  the  bacteria  present  in 
water  are  common  saprophytes  which  grow  at  the  ordinary 
temperature,  cannot  grow  at  the  temperature  of  the  body, 
and  cannot,  therefore,  produce  toxic  or  pathogenic  effects. 
The  bacteria  which  can  develop  at  the  temperature  of  the 
body  may,  or  may  not,  be  pathogenic.  This  can  only  be  de- 
cided by  an  animal  experiment.  The  method  employed  is 
as  follows: 

By  means  of  a  sterile  pipette  1  c.c.,  0.5  c.c.  and  1  drop 
of  the  water  are  added,  respectively,  to  each  of  three  tubes, 
pf  bouillon.  These  are  set  aside  in  the  incubator  at  39°  for 
24  hours.  If  no  growth  occurs  at  this  temperature  it  is  at 
once  sufficient  evidence  that  the  water  is  free  from  disease- 
producing  organisms.  On  the  other  hand,  if  a  growth  does 
develop,  injections  of  1  c.c.  of  the  culture  are  made  intra- 


440  BACTERIOLOGY. 

peritoneally  into  white  rats  or  guinea-pigs  by  means  of  a 
sterile  syringe.  The  recovery  of  the  animal  indicates  the 
absence  of  pathogenic  bacteria. 

If  death  occurs  it  may  be  due  to  the  typhoid  fever  bacil- 
lus, but  as  a  rule,  it  is  due  to  other  pathogenic  bacteria.  It 
is  necessary,  therefore,  to  identify,  if  possible,  the  noxious 
organism.  For  this  purpose,  gelatin  and  agar  plate  culti- 
vations are  made  from  the  heart  blood.  To  still  further 
test  the  pathogenic  action  of  the  organism  1  c.c.  of  a  pure 
culture  in  bouillon  is  injected  subcutaneously  into  guinea- 
pigs.  The  colon  bacillus  is  not  fatal  under  these  condi- 
tions. If  death  does  result  it  is  frequently  due  to  typhoid- 
like  bacteria  and  the  presence  of  such  organisms  should  at 
once  condemn  the  water.  The  addition  of  even  a  fraction 
of  a  drop  of  a  virulent  bouillon  typhoid  culture  to  a  liter  of 
water  can  be  detected  in  this  way. 

Aerogenic  bacteria,  such  as  the  colon  bacillus,  can  be  readily  de- 
tected by  employing"  a  fermentation  tube.  Several  forms  have  been 
devised  but  that  of  Einhorn  will  be  found  very  convenient.  The  tube 
is  filled  with  glucose  bouillon,  sterilized  and  inoculated  with  the  sus- 
pected water.  If  the  colon  bacillus  or  other  aerogenic  org-anism  is 
present,  g-as  will  be  given  off  and  will  accumulate  in  the  closed  tube. 
The  amount  of  carbonic  acid  in  this  gas  can  be  roug-hly  determined  by 
filling  the  tube  with  2  per  cent.  NaOH.  On  carefully  shaking-  the  con- 
tents of  the  tube,  the  g^as  is  brought  into  contact  with  the  alkali. 
The  difference  in  the  volume  of  the  g^as,  after  absorption,  is  due  to 
carbonic  acid.  The  residual  gas  can  be  tested,  qualitatively,  for  hy- 
drogen. For  this  purpose  the  tube  is  partially  inverted  to  allow  the 
gas  to  pass  into  the  bulb  portion.  A  lighted  match  introduced  into 
the  tube  will  cause  a  slight  explosion  (Smith). 

The  bacteriological  examination  of  snow  or  ice  is  of 
scientific  and,  at  times,  of  practical  interest.  The  material 
is  melted  in  a  sterile  dish  or  flask  and  the  water,  thus  ob- 
tained, is  examined  as  above. 

The  air  contains  a  large  number  of  bacteria  as  dry, 
finely  divided,  suspended  matter  or  dust.  The  precipita- 
tion of  rain  or  snow  mechanically  drags  down  a  considera- 


EXAMINATION    OP    WATER.  441 

ble  number  of  these  bacteria.  Consequently,  the  air  is 
purified  to  a  marked  extent  by  washing,  as  it  were,  with 
rain  or  snow. 

The  melted  water  from  freshly  fallen  snow,  collected  at 
the  ordinary  low  altitude,  may  contain  from  a  few  to  as 
many  as  500  bacteria  per  c.c.  In  the  higher  altitudes,  the 
air  contains  only  a  few  organisms  and  hence  the  precipita- 
tion in  such  places  will  contain  a  smaller  number  than  in 
the  previous  case.  The  snow  deposited  at  an  altitude  of 
6,000  feet  is  not  sterile,  but  very  nearly  so.  Usually  less 
than  five  bacteria  per  c.c.  of  melted  snow  are  found.  The 
rain-water  at  Paris  has  been  found  to  contain  from  5  to  20 
bacteria  per  c.c. 

It  follows  therefore  that  all  surface  waters,  beginning 
even  with  the  glacier  brook,  will  contain  bacteria.  As  the 
temperature  of  the  water  and  the  amount  of  organic  matter 
increases,  multiplication  of  the  bacteria  will  take  place. 
The  number  is  increased  by  contact  with  the  soil,  dust- 
laden  winds  and  above  all  by  animal  excreta  and  waste- 
matter. 

The  lakes  in  mountainous  countries,  as  in  Switzerland, 
because  of  their  high  altitude  and  the  source  of  their  water 
contain  relatively  very  few  bacteria.  The  surface  water  of 
these  lakes  usually  contains  less  than  50  and  only  occasion- 
ally over  100  bacteria  per  c.c.  Water,  however,  taken  at 
some  depth  below  the  surface  of  these  lakes  may  contain 
as  many  as  600  bacteria  per  c.c.  This  is  undoubtedly  due 
to  a  gradual  sedimentation  of  the  suspended  organisms. 

In  some  lakes,  especially  at  low  altitudes,  this  is  not 
always  the  case  and,  indeed,  the  conditions  may  be  reversed. 
.Thus,  the  surface  water  may  contain  thousands  of  bacteria 
per  c.c.,  whereas  that  near  the  bottom  may  contain  but  a 
few  hundred.  This  difference  may  be  due  to  the  fact  that 
these  lakes  are  fed  by  deep  springs;  and,  hence,  while  the 
water  at  the  bottom  is  very  cold,  that  on  the  surface  is 
warmed  by  the  sun  and  offers  conditions  which  are  favor- 


442  BACTERIOLOGY. 

v 

able  to  the  growth  of  bacteria.  In  a  given  time,  a  much 
larger  number  of  bacteria  will  be  formed  on  the  surface  by 
multiplication  then  will  be  deposited  by  sedimentation. 
Moreover,  the  currents  of  the  cold  spring-- water  from  be- 
low will  tend  to  keep  the  suspended  matter  in  the  surface 
layers. 

The  mountain  lakes,  on  the  other  hand,  are  primarily 
fed  by  the  cold,  crystal-clear  water  derived  from  glacier 
streams.  The  water,  whether  on  or  below  the  sur- 
face, has  a  minimum  low  temperature,  and,  moreover,  the 
rapid  onward  and  downward  now  prevents  the  warming  of 
that  on  the  surface.  Sedimentation  in  such  cases  may 
occur,  whereas  the  reverse,  so  far  as  numbers  are  con- 
cerned, will  be  met  with  at  lower  altitudes. 

River  waters,  according  to  the  conditions  mentioned 
above,  will  necessarily  vary  greatly  in  the  number  of  bac- 
teria which  they  contain.  Thus,  the  river  Seine  as  it  enters 
Paris  has  only  about  300  bacteria  in  a  c.c.,  whereas  when 
it  reache's  its  suburb  St.  Denis,  after  receiving  the  sewage 
of  Paris,  it  contains  200,000  per  c.c.  This  number  in  itself 
is  small  owing  to  the  rapid  flow  of  the  water  and  the  con- 
sequent enormous  dilution  of  the  sewage.  A  low  altitude, 
warm  climate,  abundance  of  organic  matter  and  a  slow  cur- 
rent will  necessarily  favor  the  multiplication  of  bacteria  in 
such  river-water.  The  number  of  bacteria  in  river-water 
under  these  conditions  may  easily  rise  to  100,000  bacteria 
or  more  per  c.c.  The  water  of  rapidly  flowing  rivers  may, 
as  a  rule,  be  said  to  contain  less  than  500  bacteria  in  a  c.c. 

The  ice  formed  on  rivers  and  lakes  will  necessarily  con- 
tain, like  the  water  itself,  a  variable  number  of  bacteria. 
The  surface  "snow-ice"  will  always  contain  a  larger  num- 
ber than  what  is  found  in  the  clear  ice.  Thus,  Prudden 
found  the  clear  ice  obtained  from  the  Hudson  river,  a  few 
miles  below  Albany,  to  yield  398  bacteria;  whereas,  the 
snow-ice  gave  9,187  bacteria  per  c.c.  of  the  water  obtained 


EXAMINATION   OF  WATER.  443- 

by  melting-  the  ice.     The  ice  supplied  in  cities,  when  melted, 
may  contain  as  many  as  25,000  bacteria  per  c.c.  of  water. 

The  rain-water  and  snow  bring1  down  from  the  air  a 
larg-e  number  of  org-anisms.  The  water  as  it  penetrates  the 
ground  is  filtered,  and,  in  this  way,  all  the  bacteria  are  re- 
tained by  the  upper  layer  of  the  soil.  It  follows,  therefore, 
that  water  coming-  from  the  deeper  layers  of  the  earth  is 
practically  g-erm-free.  This  is  often  the  case  with  spring-- 
water. As  a  rule,  however,  a  small  number  of  bacteria 
from  the  surface  soil  re-enter  the  water  as  it  reaches  the 
surface.  Hence,  spring"-water  usually  contains  less  than  50 
bacteria  per  c.c. 

The  water  of  artesian  or  tubular  wells,  for  reasons  just 
given,  will  likewise  be  free  from  bacteria.  A  small  numbej 
of  these  are  usually  present,  but  this  is  due  to  contamina- 
tion at  or  near  the  surface. 

Ordinary  wells,  as  migiit  be  expected,  give  the  great- 
est known  variation  in  the  number  of  bacteria.  The  well 
water  may  contain  practically  no  bacteria  and  on  the  other 
hand  they  may  be  almost  innumerable.  Thus,  a  number  of 
wells  have  been  found  to  contain  as  many  as  800,000  bac- 
teria per  c.c.  As  a  rule,  a  well-water,  especially  when  it 
is  very  cold,  will  contain  about  1,000  bacteria  per  c.c. 

In  sea-water,  there  is  less  variation  in  the  number  and 
kind  of  bacteria  present,  as  well  as  in  their  distribution 
from  the  shore  or  from  the  bottom  than  is  usually  met  with 
in  ordinary  waters.  Russell  has  shown  that  the  ocean  sur- 
face water  contains  from  a  few  to  as  many  as  120  bacteria 
per  c.c.  This  number,  however,  may  at  times  be  consider- 
ably increased  and  may  even  reach  28,000.  In  the  deeper 
layers  of  the  water  they  are  no  more  abundant  than  on  the 
surface.  The  slime  at  the  bottom  of  the  sea,  at  Naples, 
contained  about  300,000  while  at  Wood's  Holl  only  about 
17,000  bacteria  were  present  in  a  c.c.  The  enormous  differ- 
ence between  the  number  of  bacteria  in  the  slime  and  in  the 


444  BACTERIOLOGY. 

overlying  water  is  largely  due  to  the  fact  that  certain  bac- 
teria can  live  and  multiply  in  the  slime.  Fully  one-third  of 
the  bacteria  present  in  the  slime  belonged  to  three  species 
which  were  met  with  only  in  this  material.  Certain  species 
of  slime  bacteria  found  at  Naples  at  a  depth  of  3,500  feet 
were  also  met  with  at  Wood's  Holl,  near  the  coast  as  well  as 
at  a  distance  of  100  miles.  It  is  of  interest  to  note  that 
Fischer  obtained  no  bacteria  from  slime  gathered  at  a  depth 
of  1-3  miles. 

Sewage  water,  necessarily,  is  very  rich  in  bacteria. 
Usually  a  drop  will  be  found  to  contain  several  hundred 
thousand.  The  sewage  water  in  large  cities  like  Paris  and 
London  is  known  to  contain  as  many  as  6  or  8  millions  of 
bacteria  per  c.c.  The  water  in  the  public  washing  stations 
which  float  in  the  Seine  at  Paris  was  found  to  contain  from 
12  to  40  millions  of  bacteria  in  a  c.c. 


Soil. 


The  upper  layers  of  the  soil  contain  enormous  numbers 
of  bacteria  which  play  a  most  important  part  in  the  econ- 
omy of  nature.  As  indicated  in  Chapter  V  (p.  112),  the  dead 
animal  and  vegetable  matter  deposited  upon  the  surface  of 
the  earth  is  sooner  or  later  broken  up  into  the  simplest  of 
inorganic  compounds, — carbonic  acid,  water,  ammonia, 
nitrous  and  nitric  acids,  hydrogen  sulphide,  etc.  This  is 
done  through  the  agency  of  the  bacteria  in  the  soil.  With- 
out their  presence  and  activity,  the  dead  matter  would  re- 
main as  such,  and  would  accumulate  as  layer  on  layer. 
The  nitrogen  of  the  protein  matter  which  is  contained  in  the 
protoplasm  of  animal  and  plant  cells  is  derived  from  the  in- 
organic nitrogen  of  the  soil.  Were  it  not  for  the  bacterial 
activity  carried  on  in  the  soil  this  supply  of  inorganic  nitro- 
gen would  in  time  become  exhausted,  and,  as  pointed  out  by 
Pasteur,  life  would  soon  cease  to  exist.  The  dead  plant  and 


EXAMINATION   OF    SOIL.  44fr 

animal,  composed  of  the  most  complex  chemical  compounds, 
serve  as  food  for  this  microscopic  world  which  in  turn  con- 
verts the  elements  present  into  compounds  which  are  ^ui%— 
able  for  the  maintenance  of  higher  plant,  and  thus  of  higher 
animal  life. 

The  bacterial  changes  going  on  in  the  soil  are  usually 
designated  as  those,  of  fermentation  or  putrefaction.  Either 
term  implies  the  tearing  down  by  bacteria  or  other  organ- 
isms, of  complex  matter  and  transforming  this  into  simpler 
forms.  While  a  small  number  of  bacteria  may  be  consid- 
ered as  the  most  relentless  foes  of  man,  animals  and  even 
of  plants,  yet  the  vast  army  of  these  organisms  constitute 
man's  best  friend. 

The  changes  induced  by  bacteria,  as  usually  observed, 
are  analytic  or  reducing  in  character.  Some  of  the  organ- 
isms in  the  soil,  however,  give  rise  to  important  oxidation 
changes,  such  as  is  seen  in  the  conversion  of  ammonia  into 
nitrous  and  nitric  acids.  The  so-called  nitrifying  bacteria 
are  found  widely  distributed  in  the  soil  and  in  water.  Under 
certain  special  conditions  their  action  is  so  pronounced  as 
to  give  rise  to  vast  quantities  of  their  characteristic  pro- 
duct. The  salt-peter  of  India  or  potassium  nitrate,  and  the 
Chili  salt-peter  or  sodium  nitrate,  as  indicated  heretofore, 
are  most  valuable  commercial  products  which  result  from 
the  action  of  certain  bacteria  upon  animal  excreta. 

Another  interesting  group  of  soil  bacteria,  already  re- 
ferred to  (p.  110),  is  met  with  in  the  characteristic  nodules 
on  the  roots  of  leguminous  plants.  These  bacteria  have 
undoubtedly  the  power  of  assimilating  the  free  nitrogen  of 
the  air  and  of  transmitting  it  to  the  growing  plant. 
Strange  to  say,  the  higher  plant  in  this  case  is  practically 
dependent  upon  these  parasitic  bacteria  for  its  existence. 
It  may  grow  in  sterile  soil  but  the  growth  in  that  case  is 
poor  and  dwarfed,  and  presents  a  striking  contrast  to  the 
plant  growing  in  soil  to  which  these  organisms  have  been, 
added. 


446  BACTERIOLOGY. 

The  soil  may  be  considered  as  the  natural  habitat  of 
certain  pathogenic  bacteria,  notably, — tetanus,  malignant 
edema,  anthrax,  pus-producing-  cocci  and  symptomatic 
anthrax.  The  latter  has  not  been  isolated  from  the  soil 
but  the  others  have  been  repeatedly  found  there.  This 
is  true  especially  of  tetanus  and  of  malignant  edema 
which  seem  to  be  distributed  in  the  soil  over  the  entire 
surface  of  the  globe. 

Owing"  to  the  enormous  numbers  of  common  saprophy tic  bacteria, 
the  tetanus  or  malignant  edema  bacilli  cannot  be  isolated  direct 
from  the  soil,  by  the  ordinary  plate  method.  It  is  necessary  to  resort 
to  an  animal  experiment  in  order  to  effect  their  isolation.  For  this 
purpose,  an  incision  is  made  through  the  skin  of  a  rabbit  or  guinea- 
pig-  and  a  small  pocket  is  made  into  the  subcutaneous  tissue  (see 
p.  262).  A  small  amount  of  the  suspected  soil  is  then  introduced  into 
this  pouch.  Most  of  the  common  bacteria  are  unable  to  grow  in  the 
body  and  are  soon  destroyed.  The  tetanus  or  malignant  edema  germ, 
if  present,  is  favored  by  these  saprophytic  bacteria  and  is  thus  enabled 
to  multiply  and  to  produce  the  poison  which  soon  destroys  the  animal. 
From  the  local  abscess  or  from  the  tissues  and  serous  surfaces  of  the 
animal,  anaerobic  cultures  (p.  311)  can  then  be  made  and  the  organ- 
ism isolated  in  pure  culture.  » 

The  above  method  is  usually  employed  when  attempting  to 
isolate  the  organisms  from  the  pus  of  a  wound  in  a  case  of  the  dis- 
•ease.  The  direct  isolation  of  the  tetanus  bacillus  by  the  plate 
method  was  accomplished  once,  in  this  laboratory,  from  a  case  which 
originated  by  infection  from  a  tooth. 

The  typhoid  bacillus,  as  mentioned  on  p.  427,  has  been  isolated 
from  the  soil.  The  cholera  vibrio  may  at  times  occur  in  the  soil,  but 
.as  yet  it  has  not  been  found  there.  A  non-virulent  and  even  a  viru- 
lent pest  bacillus  has  been  found  in  the  earth. 

Some  idea  of  the  enormous  number  of  bacteria  present 
may  be  given  when  it  is  said  that  1  c.c.  of  the  surface 
•soil  usually  contains  several  hundred  thousand  bacteria. 
Indeed  some  observers  have  reported  as  many  as  50  to  80 
millions  of  these  organisms. 

The  rain  and  snow  bring  down  a  great  many  bacteria 
from  the  air  but,  as  soon  as  the  soil  dries  and  becomes  pul- 


EXAMINATION   OF    SOIL.  447 

verized  to  dust,  these  and  many  others  return  to  the  atmo- 
sphere as  a  result  of  the  action  of  wind  or  of  other  agencies. 
The  bacteria  brought  down  from  the  air  do  not  penetrate 
the  deeper  layers  of  the  soil.  They  are  retained  in  the 
surface  layers  inasmuch  as  the  earth  is  a  good  mechanical 
filter.  For  this  reason,  the  number  of  bacteria  in  the  earth 
diminishes  with  the  depth.  At  a  depth  of  about  6  feet  the 
number  of  bacteria  has  greatly  decreased.  Sometimes  the 
soil  will  be  sterile;  at  other  times,  only  a  few  hundred  bac- 
teria will  be  present.  At  a  depth  of  9  to  12  feet  the  soil  is 
practically  sterile.  Not  only  is  it  impossible  for  the  bacteria 
to  penetrate  from  the  surface  downwards,  for  any  great 
distance,  but  it  is  also  impossible  for  many  organisms  to 
survive  for  any  length  of  time  under  such  conditions.  This 
fact  was  unconsciously  recognized  by  man,  ages  ago.  Thus, 
in  times  of  epidemics,  as  that  of  the  Black  Plague  in 
London,  in  1665,  bodies  were  ordered  to  be  buried  at  a  depth 
of  not  less  than  6  feet. 

Burial  experiments  made  with  cadavers  of  hogs  and 
other  animals,  into  which  large  quantities  of  pure  cultures 
were  injected  or  in  which  diseased  tissue  was  placed,  have 
shown  that  at  a  depth  of  3  to  4^  feet  many  bacteria  are 
destroyed  within  a  month.  This  was  the  case  with  pure 
cultures  of  the  typhoid,  cholera  and  Friedliinder  germs. 
In  a  typhoid  spleen  the  Eberth  bacillus  was  found  alive  on 
the  96th  day.  The  tubercle  bacillus  was  alive  and  virulent 
on  the  95th  day  but  was  dead  in  123  days.  The  tetanus 
spores  died  out  between  the  8th  and  12th  month,  and 
anthrax  spores  remained  virulent  at  the  end  of  11  months. 
The  hog  erysipelas  bacillus  was  alive  after  8  months.  The 
bacillus  of  green  pus  and  the  Micrococcus  tetragenus  died 
between  the  1st  and  4th  month.  In  only  two  instances 
could  the  pathogenic  germ  be  detected  outside  of  the 
cadaver.  The  disease-producing  organisms,  present  in  a 
cadaver,  are  destroyed  in  time  when  buried  in  the  soil. 


448  BACTERIOLOGY. 

Moreover,  the  buried  organism,  owing"  to  the  filtering-  power 
of  the  soil,  is  not  to  be  considered  as  a  source  of  danger, 
especially  if  surrounded  by  dry  earth. 

Method  of  analysis. — The  collection  of  samples  of  earth 
from  various  depths  can  be  readily  accomplished  by  means 
of  Praenkel's  earth-borer.  For  each  culture  experiment  a 
definite  quantity  of  the  soil  should  be  weighed  out,  or  a 
measured  volume  taken.  The  latter  is  the  simpler  proce- 
dure, and  can  be  done  with  a  small  platinum  spoon  which 
has  a  capacity  of  ^V  c.c. 


FIG.  59.    Esmarch's  apparatus  for  counting  colonies  in  roll-tubes. 

With  the  above  sterile  instrument  one  spoonful  of  the 
earth  is  transferred  to  a  tube  of  liquid  gelatin.  The  con- 
tents of  the  tube  are  then  mixed  thoroughly,  with  a  steril- 
ized platinum  wire,  and  an  Esmarch  roll-tube  is  made.  The 
soil  and  the  organisms  present  are  thus  brought  into  per- 
fect contact  with  the  gelatin,  and  after  a  lapse  of  a  few 
days  colonies  develop. 

The  colonies  can  be  readily  counted,  either  direct,  or  by  the  aid 
of  an  Esmarch  roll-tube  counter.  This  apparatus  is  shown  in  Fig-.  59. 
The  roll- tube  is  placed  in  the  holder  and  the  average  number  of 


EXAMINATION   OF   AIR.  449 

colonies  in  a  square  cm.  is  ascertained  in  the  same  manner  as  de- 
scribed under  water  analysis.  The  area  of  the  gelatin  cylinder  is 
then  calculated  from  the  formula  TT  D  a;  in  which  TT  is  3.1416,  D  the 
diameter  of  the  tube  and  a  the  length  of  the  gelatin  cylinder.  The 
total  number  of  colonies  can  then  be  determined.  The  kind  of  organ- 
isms present— bacteria,  moulds,  etc.— can  be  determined  by  a  study 
of  the  colonies,  and  by  further  culture  and  examination. 

In  this  way  it  is  easy  to  determine  approximately  the 
number  and  kind  of  organisms  present.  Unfortunately, 
this  method  is  not  adapted  for  the  detection  of  anaerobic 
bacteria  which  are  apparently  widely  distributed  in  the 
earth,  and  are  represented  by  the  well-known  bacilli  of 
tetanus,  malignant  edema  and  symptomatic  anthrax. 
These  have  thus  far  been  obtained  from  the  soil  only  by 
the  indirect  method  mentioned  on  p.  446. 


Air. 


The  bacteria  present  in  the  air  are  derived  almost  en- 
tirely from  the  soil.  When  the  surface  of  the  earth  be- 
comes dry,  the  fine  particles  of  dust  afe  readily  taken  up 
by  gusts  of  wind  and  may  then  be  carried  upward  to  a  con- 
siderable altitude.  To  a  slight  extent,  germs  may  enter 
the  air  from  the  water.  This  can  happen,  however,  only 
when  the  water  is  violently  thrown  into  a  spray  as  in  the 
neighborhood  of  a  water-fall  or  in  the  case  of  storm-lashed 
waves.  The  extremely  fine  particles  of  water  are  carried 
into  the  air  and  eventually  may  dry  up  leaving  the  solid 
organisms  behind. 

It  should  be  clearly  understood  that  germs  do  not  and 
cannot  enter  the  air  of  themselves.  They  must  always  be 
torn  by  force  from  the  soil  or  from  the  water.  Moreover, 
what  is  equally  important,  they  are  not  removed  from  moist 
surfaces  or  from  water  by  ordinary  currents  of  air.  The 
most  pathogenic  organism  growing  on  the  ordinary  culture 

29 


450  BACTERIOLOGY. 

media  cannot  leave  the  moist  surface.  For  the  same  rea- 
son, the  breath  of  a  person  is  practically  free  from  bac- 
teria; or,  at  all  events,  it  does  not  take  up  and  carry  out 
the  organisms  that  may  be  present  in  the  air-passages. 
The  breath  of  a  consumptive,  therefore,  is  free  from  the 
specific  germ  of  this  disease.  It  is  only  in  case  of  violent 
expulsion  of  air,  as  in  a  fit  of  coughing,  that  particles  of 
moisture  with  the  contained  organisms  may  be  forcibly 
ejected  from  the  air  passages. 

When  air  is  passed  over  a  moist  surface,  instead  of 
taking  up  bacteria,  it  is  usually  deprived  of  those  that  are 
present.  The  suspended  particles,  whether  mere  grains  of 
dust  or  the  much  smaller  bacterial  cells,  when  once  brought 
into  contact  with  a  moist  surface  become  fixed  to  that  sur- 
face. Hence  it  is,  that  the  farther  one  goes  away  from  the 
shore  the  less  numerous  the  air  germs  become.  The  air 
at  sea,  at  some  distance  from  the  land,  may  be  said  to  be 
practically  free  from  organisms. 

The  suspended  particles  in  the  air  are  made  up,  in  the 
first  place,  of  the  relatively  heavy  grains  of  dust.  The 
surface  of  these  dust  particles  is  covered  with  such  organ- 
isms as  chanced  to  dry  down.  To  a  less  extent  the  sun 
motes  can  be  considered  as  the  carriers  of  bacteria.  The 
free  organism,  either  single  or,  more  often  in  short 
threads  or  groups,  constitutes  the  finest  particle  in  the 
air. 

The  atmospheric  germs,  by  no  means,  belong  exclu- 
sively to  the  group  of  bacteria.  While  this  form  is  quite 
common,  yet  the  yeast  is  frequently  met  with  and  the  spores 
of  moulds  are  very  abundant.  Inasmuch  as  these  several 
forms  of  living  matter  exist  in  the  air  as  dried  particles  it 
is  evident  that  they  do  not  multiply.  On  the  contrary, 
desiccation,  when  prolonged,  tends  to  destroy  many  organ- 
isms and  this  unfavorable  action  is  still  further  accentuated 
by  the  direct  germicidal  effect  of  sun-light. 


EXAMINATION   OF   AIR.  451 

The  particles  of  dust  and  the  free  organisms  suspended 
in  the  atmosphere  are  specifically  heavier  than  the  air  and 
hence  tend  to  settle.  This  sedimentation  is  very  marked 
even  in  a  few  hours,  in  a  room  where  currents  of  air  are 
absent.  To  a  certain  extent  this  takes  place  in  the  open 
air,  but  the  purification  of  the  out-door  atmosphere  is  chiefly 
accomplished  by  the  precipitation  of  rain  and  snow,  and  by 
the  washing-  of  the  air  as  it  passes  over  the  surface  of  lakes 
and  seas.  For  the  same  reason  expired  air  will  contain  but 
a  very  small  fraction  of  the  organisms  present  in  that  which 
was  inspired.  Thus,  when  the  former  contained  20,000 
germs  per  cubic  meter,  the  latter  contained  in  a  like  volume 
only  40.  It  is  evident,  therefore,  that  air  which  contains 
the  fewest  organisms  is  to  be  met  with  at  high  altitudes,  as 
on  the  tops  of  mountains  and  glaciers,  and  in  mid-ocean. 

The  actual  number  of  organisms  present  in  the  air  is 
greatly  over-estimated.  This  is  indicated  in  the  marked 
freedom  from  contamination  by  air  germs,  of  the  nutrient 
media,  in  ordinary  routine  bacteriological  work.  Naturally, 
the  air  of  a  recently  swept  room  will  contain  more  germs 
than  that  outside  of  the  house.  Likewise,  the  air  of  cities 
will  be  relatively  rich  as  compared  with  that  of  the  open 
country.  Moreover,  the  number  of  organisms  in  the  air 
during  the  winter  season  will  be  less,  nearly  one-half,  than 
in  the  spring  or  summer. 

The  following  table  will  be  of  interest  showing  the 
variation  in  the  number  of  bacteria  and  moulds  in  different 
seasons  and  in  country  and  city  air.  The  figures  represent 
the  average  numbers  obtained  by  Miquel,  who  made  monthly 
examinations  extending  over  a  period  of  10  years.  The  re- 
sults in  the  first  half  of  the  table  were  obtained  at  the 
Mont-souris  observatory,  situated  in  a  park  at  the  southern 
edge  of  Paris;  whereas,  the  results  in  the  other  half  of  the. 
table  were  obtained  in  the  square  before  the  City  Hall,  only 
about  2  miles  distant  from  the  former  place.  The  figures 


452 


BACTERIOLOGY. 


give   the  number  of  organisms  present  in  a   cubic   meter 
(1000  liters)  of  air. 


ji 

MONT-SOURIS. 

CITY   HALL. 

BACTERIA. 

MOULDS. 

BACTERIA. 

MOULDS. 

Winter  

170 

295 
345 
195 

145 

195 
245 
230 

4305 

8080 
9845 
5665 

1345 
2275 
2500 
2185 

SDrin«r 

Summer  

Autumn. 

Average 

250 

205 

6975 

2705 

On  an  average,  it  may  be  said  that  the  country  air 
contains  from  1  to  5  germs  in  10  liters.  Not  infrequently 
the  air  of  private  rooms  and  hospitals,  especially  after 
sweeping,  may  contain  20  to  50  thousand  organisms.  In 
the  absence  of  currents  of  air  these  suspended  germs  rapidly 
settle  and  are  not  removed  by  ordinary  ventilation.  Dry 
sweeping  and  dusting  should,  therefore,  be  avoided,  especially 
in  rooms  which  are  occupied  by  persons  afflicted  with  con- 
sumpton  or  with  other  infectious  diseases. 

The  vast  majority  of  the  bacteria  present  in  the  air  are 
harmless  saprophytes.  Pathogenic  bacteria  are  present, 
relatively,  in  very  small  numbers  and  hence  their  detection 
is  extremely  difficult.  The  pus-producing  micrococci  have 
been  isolated  direct  from  the  air.  The  tubercle  bacillus 
enters  the  body  through  the  air  and  yet  direct  examinations, 
for  the  reasons  given,  have  thus  far  been  negative.  It  has 
been  detected,  however,  in  the  dust  of  rooms, — that  is  to 
say  in  the  sediment  deposited  from  the  air. 

Method  of  analysis. — It  was  pointed  out  in  connection 
with  the  analysis  of  water  (p.  437)  that  the  number  of  bac- 
teria present  could  not  be  determined  absolutely.  The  same 


EXAMINATION    OF    AIR.  453 

is  true  of  the  various  methods  employed  in  the  determina- 
tion of  the  number  of  atmospheric  germs.  If,  for  instance, 
all  the  organisms  present  in  a  given  volume  of  air  are  trans- 
ferred to  water  it  does  not  follow  that  they  will  all  develop 
upon  the  gelatin  plate  which  is  made  from  such  water. 
Many  of  these  may  be  anaerobic  germs,  others  may  require 
the  temperature  of  the  body,  and  again  a  large  number  may 
fail  to  grow  because  the  medium  is  not  a  suitable  one.  Con- 
sequently, the  results  obtained  in  the  examination  of  air 
are  to  be  considered  as  merely  relative  in  value. 

The  earliest  method  of  examining-  air,  that  of  Pasteur,  consisted 
in  breaking-  open,  in  the  desired  locality,  a  sterile  flask  from  which 
the  air  had  been  previously  expelled  by  boiling-.  The  air  rushing-  into 
the  vacuum  flask  carried  with  it  the  suspended  organisms  which  then 
developed  in  the  nutrient  medium  present.  This  method  demonstrated 
that  atmospheric  germs  were  not  as  numerous  as  had  been  supposed, 
and.  furthermore,  that  they  were  not  evenly  distributed  throughout 
the  air.  Koch  endeavored  to  determine  the  number  and  kind  of  bac-* 
teria  present  in  a  given  volume  of  air  by  making-  use  of  the  gelatin 
plate  method.  Sterile  gelatin,  for  example,  was  placed  in  an  open 
Esmarch  dish  within  a  plugged  glass  cylinder  of  known  volume.  The 
apparatus  was  sterilized  and  then  taken  to  the  desired  locality  where 
the  cotton  plug  was  removed.  The  cylinder  was  now  allowed  to  re- 
main open  for  some  minutes  to  allow  the  germ-laden  air  to  enter.  The 
plug  was  then  replaced  and  the  apparatus  was  set  aside.  The  organ- 
isms present  in  this  confined  volume  of  air  soon  settled  upon  the  sur- 
face of  the  gelatin  where  they  developed  forming  colonies.  The 
method,  though  simple,  is  extremely  imperfect  and  fully  as  good  re- 
sults can  be  obtained  by  exposing  an  ordinary  gelatin  plate  direct  to 
the  air  for  10  to  30  minutes. 

Very  fair  quantitative  results  can  be  obtained  by  means  of 
Hesse's  apparatus.  This  consists  of  a  glass-tube  70  cm.  long  and  3  or 
4  cm.  in  diameter.  One  end  of  the  tube  is  closed  with  a  stopper 
through  which  passes  a  short  glass  tube.  This  is  connected  by  means 
of  rubber  tubing  with  an  aspirating  bottle  of  known  volume.  The 
other  end  of  the  tube  is  closed  with  two  rubber  caps,  the  inner  one  of 
which  has  a  small  central  opening.  The  tube  is  first  sterilized  and 
then  about  50  c.c.  of  gelatin  are  introduced.  The  gelatin  is  then  solid- 
ified in  a  thin  film  over  the  inner  wall  of  the  tube  thus  making  a  large 
Esmarch  roll- tube.  The  apparatus  is  placed  in  the  room  or  locality 


454 


BACTERIOLOGY. 


where  the  air  is  to  be  examined;  the  outer  cap  is  removed  and  the  water 
in  the  aspirating-  bottle  is  then  allowed  to  escape.  Any  desired  vol- 
ume of  air  can  thus  be  drawn  through  the  tube.  The  bacteria  pres- 
ent in  the  air,  coming-  into  contact  with  the  gelatin,  become  fixed  and 
after  the  tube  is  set  aside  they  develop.  The  colonies  can  then  be 
counted,  and,  inasmuch  as  the  volume  of  air  drawn  through  the  ap- 
paratus is  known,  the  average  number  of  germs  per  liter  of  air  can 
be  ascertained. 

A  more  exact  method,  requiring  however  very  expen- 
sive apparatus,  is  that  of  Petri.  In  this  method  the  air  is 
drawn  through  a  sterile  sand  filter 
Fig.  66  eZ,  p.  469.  The  suspended 
particles,  dust  and  organisms,  are 
held  back  by  the  well-packed,  fine 
grained  sand.  The  contents  of  the 
filter  are  then  distributed  into  a  num- 
ber of  Petri  dishes,  gelatin  is  added 
and  after  thorough  mixing  it  is  al- 
lowed to  solidify.  The  colonies  that 
now  develop  can  be  examined  and 
counted  with  the  same  ease  as  in 
water  analysis.  In  this  method  it  is 
possible  for  a  large  number  of  bac- 
teria to  adhere  to  a  single  grain  of 
sand  and  when  development  takes 
place  the  result  will  be  but  one 
colony.  The  objection  can  be  over- 
A  come,  in  part,  by  substituting  a  sol- 

FIG.  60.    Apparatus  for  exami-       .  ,  _         _, 

nation  of  air.    A—  Sedgwick-  uble  compound  for  the  sand.     Thus, 

Tucker's;  B— Straus-Wurtz's. 

powdered  sodium  sulphate,  or  better, 

cane-sugar  may  be  employed.  Filters  containing  sugar 
have  been  employed  by  Miquel,  and  by  Sedgwick  and 
Tucker  (Fig.  60  a).  On  the  addition  of  gelatin,  the  sugar 
dissolves  leaving  the  germs  present  in  a  fine  state  of 
division.  The  number  of  colonies,  thus  obtained,  repre- 
sents quite  closely  the  number  of  cells  originally  present 
in  the  air. 


a 


EXAMINATION  OF    AlR.  455 

A  third  method  of  procedure  consists  in  aspirating"  the 
air  through  sterile  water  or  gelatin.  The  bubbles  of  air  are 
thus  washed  and  the  germs  present  are  retained  by  the 
liquid.  A  very  useful  and  cheap  apparatus  for  this  purpose 
is  that  employed  by  Straus  and  Wurtz,  and  is  shown  in  Pig1. 
60  b.  It  consists  of  a  glass  cylinder  a  in  the  neck  of  which 
is  fitted  a  ground  pipette  or  tube  &,  the  upper  end  of  which 
is  plugged  with  cotton. 

The  side-arm  c  is  provided  with  two  cotton  plugs  one 
above  and  one  below  the  middle  constriction.  The  appar- 
atus is  sterilized  in  the  dry-heat  oven;  10  c.c.  of  gelatin  are 
then  introduced  and  a  drop  of  sterile  oil  is  added  to  pre- 
vent subsequent  foaming.  The  plug  is  removed  from  the 
end  of  the  pipette  &  while  the  side-tube  c  is  connected  with 
an  aspirator.  The  air  is  drawn  rapidly  through  the  gela- 
tin, and,  as  a  result,  it  is  deprived  of  most  of  the  suspended 
organisms.  The  remainder  are  held  back  by  the  cotton 
plug  on  the  inside  of  the  constriction  in  the  side-tube  c.  At 
the  close  of  the  operation  this  plug  is  pushed  down  into  the 
cylinder  and  thoroughly  agitated  with  the  gelatin  in  order 
to  bring  the  adhering  organisms  into  suspension.  The 
gelatin  can  be  solidified  on  the  inside  of  the  cylinder,  thus 
forming  an  Esmarch  roll-tube;  or,  definite  portions  of  the 
gelatin  (0.5  and  0.1  c.c.)  can  be  transferred  to  gelatin  tubes 
and  Petri  plates  can  then  be  made  as  in  water  analysis. 

Laboratory  work. — The  student  will  examine  two  samples  of 
water,  one  drawn  from  the  tap  and  the  other  from  a  well.  The  num- 
ber and  kind  of  colonies  are  to  be  reported.  A  specimen  of  milk  will 
also  be  plated  and  examined  in  the  same  manner  as  in  water  analysis. 

Esmarch  roll-tubes  will  be  made  with  three  samples  of  soil. 


CHAPTER     XIV. 

SPECIAL  METHODS  OF  WORK. 

Pipettes. 

Sterile,  drawn-out  tube  pipettes,  as  employed  by  the 
Pasteur  school,  are  invaluable  in  bacteriological  work. 
They  can  be  readily  prepared  and  a  good  supply  should  be 
kept  on  hand.  The  method  of  preparing  pipettes  is  as 
follows : 

Glass-tubing  having  a  diameter  of  about  8  mm.  is  cut 
up  into  lengths  of  about  35  cm.  (14  in.).  A  slight  constric- 
tion is  made  at  a  distance  of  about  6  cm.  (2^  in.)  from  e'ach 
end.  This  is  not  necessary,  but  it  serves  to  prevent  the 
cotton  plug  from  descending.  Moreover,  the  tube  can  be 
readily  sealed  at  this  place  whenever  it  is  desirable  to  do 
so.  The  ends  are  then  rounded  in  the  blast-lamp.  A  piece 
of  cotton  is  pushed  into  each  end  of  the  tube  by  means  of  a 
drawn-out  piece  of  glass  tubing.  The  tubes,  thus  equipped 
(Fig.  61  a)  are  placed  in  a  horizontal  position  in  a  dry-heat 
oven  and  sterilized  (p.  160).  The  sterilized  tubes  should  be 
kept  in  stock  and  from  these  the  pipettes  can  be  made  in  a 
few  minutes  whenever  desired. 

To  make  the  pipette  proper,  the  plugged  sterile  tube 
(a)  is  heated  at  the  middle  in  a  blast-lamp.  The  broad 
flame  should  be  used  in  order  to  soften  as  long  a  piece  of 
the  tubing  as  possible  (3-5  cm.).  When  the  middle  is  thor- 
oughly softened  the  two  halves  should  be  slowly  drawn 
apart.  A  relatively  wide,  thick-walled  capillary  is  thus 
obtained;  whereas,  if  the  tube  is  drawn  out  rapidly  the  re- 
sulting capillary  will  be  very  narrow  and  thin  walled.  The 


PIPETTES.  457 

drawn-out  portion  should  be  about  40  cm.  (16  in.)  long. 
This  is  then  heated  in  the  middle,  and,  as  a  result,  two 
sealed  pipettes  are  thus  obtained  (Fig.  61,  c  or  d). 

Frequently,  it  is  desirable  to  transfer  a  liquid  or  a  sus- 
pension from  one  tube  to  another.  For  small  quantities 
the  ordinary  pipette  (c  or  d)  can  be  used  direct.  For  larger 
quantities  a  bulb  should  be  blown  into  pipettes  c  or  d,  thus 
giving  pipette  d.  While  it  requires  a  great  deal  of  practice 
to  blow  a  perfect  bulb,  still  a  serviceable  one  can  be  made 
without  much  difficulty. 


a 
4 


FIG.  Gi.  Drawn-out  tube  pipettes  of  Pasteur,  a — Plugged,  sterile  tube  as  kept  in 
stock;  b— The  same  heated 'at  x  in  blast-lamp,  drawn  out;  then  sealed  at  x\  c  and  ^—com- 
pleted pipettes;  e — The  same  with  bulb. 

A  narrow  flame  of  the  blast-lamp  is  directed  at  the  tube  a  short 
distance  above  the  point  where  it  begins  to  narrow.  The  tube  is 
slowly  turned  until  the  glass  softens;  whereupon  the  ends  are  slightly 
pushed  together  so  as  to  form  a  thick  ring  of  glass.  One  or  two  addi- 
tional rings  are  made  in  this  way.  A  large  flame  is  then  turned  on 
so  as  to  melt  the  thickened  zones  of  glass.  When  the  glass  is  per- 
fectly soft  the  end  is  brought  rapidly  to  the  mouth  and  the  bulb  is 
blown.  The  glass  tube  should  be  turned,  slowly  and  steadily,  while 
heating  and  also  while  the  bulb  is  being  blown.  The  bulb  pipettes 
can  often  be  used  to  better  advantage  if  the  capillary  is  bent  at  right 
angles  just  below  the  bulb. 

When  a  pipette  is  to  be  used  the  mouth  end  should  be  heated  in 
the  flame  for  a  few  moments.  This  is  to  prevent  possible  infection. 
The  lower  end  of  the  capillary  tube  is  then  scratched  with  a  file  or 
glass-cutting  knife  and  the  sealed  end  removed.  The  end  may  be 


458  BACTERIOLOGY. 

broken  off  by  slightly  bending-  it  between  the  fingers.  The  open  capil- 
lary end  should  now  be  passed  several  times  through  the  flame  in 
order  to  sterilize  the  exterior.  By  blowing  through  the  tube,  direct- 
ing the  stream  of  air  against  the  back  of  the  hand,  one  can  ascertain 
when  the  tube  has  cooled  sufficiently  for  use.  The  pipette  is  then  in- 
serted into  the  test-tube  or  other  container  and  the  liquid  is  drawn 
up  by  suction.  The  end  of  the  tube  in  the  mouth  is  closed  by  the 
tongue  or  finger,  and  the  pipette  is  then  withdrawn.  The  liquid  can 
now  be  transferred  to  another  tube  or  to  a  series  of  tubes  or  flasks* 

The  pipette  is  very  useful  in  removing1  the  contents  of 
a  pus  cavity  in  an  animal,  in  collecting-  an  exudate  (p.  277) 
or  in  drawing  blood  from  the  heart  (p.  279).  It  can  be  used 
to  remove  liquid  cultures  or  surface  cultures  from  agar,. 
potato,  etc.  In  the  latter  case,  sterile  water  or  bouillon  is 
transferred  to  the  tube  by  means  of  the  pipette.  The 
growth  is  whipped  up  to  make  a  fine  suspension  which 
then  can  be  drawn  up  into  the  pipette.  The  pipette  is  ex- 
tremely useful  when  it  is  desirable  to  inoculate  a  large 
number  of  tubes;  in  the  tests  for  agglutination  and  in  the 
preparation  of  sacs.  It  is  even  more  useful  than  the  plati- 
num wire. 

The  material  can  be  kept  in  the  tube  by  sealing-  the 
capillary  end.  If  at  the  same  time  the  tube  is  sealed  below 
the  cotton  plug,  the  culture  or  material  can  thus  be  kept 
perfectly  free  from  contamination  or  desiccation.  It  is  fre- 
quently desirable  to  preserve  the  heart-blood  of  an  animal, 
and  thus  keep,  the  org-anism  that  may  be  present  in  its  full 
virulence.  This  may  be  done  in  the  manner  described  or 
by  the  following  slig-htly  modified  procedure. 

A  pipette  is  heated  about  a  half  an  inch  beyond  where 
it  begins  to  narrow.  When  the  glass  has  softened  it  is 
drawn  out  about  15  cm.  (6  in.).  Fig.  62  a  shows  such  a 
pipette.  The  bulb  and  capillary  is  then  filled  to  the  x 
mark  according  to  the  directions  given  above.  The  capil- 
lary is  then  sealed  at  the  end  and  also  at  the  x  mark.  The 
growth  or  blood  is  thus  sealed  in  a  bulb  tube  about  15  cm. 


PIPETTES. 


459 


long1.     The  same  pipette  can  be  utilized  for  preparing1  a 
number  of  such  sealed  tubes. 

In  testing  the  action  of  moist  heat  on  bacteria  it  is  advisable  to 
draw  up  the  liquid  into  the  capillary  to  a  height  of  8-10  cm.  (3-4  in.) 
as  shown  in  Fig.  62  c.  The  capillary  in  then  sealed  at  the  end  and  at 
x  just  above  the  liquid.  The  used  pipette  can  then  be  drawn  out 
again  as  in  Fig.  62  a.  By  cutting  off  the  tube  above  the  bulb  portion, 
the  capillary  portion  can  be  filled  and  sealed  in  the  manner  just 
described.  The  same  pipette  can  thus  be  used  for  the  preparation  of 
a  large  number  of  sealed  capillary  tubes.  When  it  is  desired  to  re- 
move the  liquid  from  the  narrow,  sealed,  capillary  tube,  one  end  should 
be  cut  off  and  sterilized  by  naming.  This  end  is  then  introduced  into 
the  culture  tube  which  is  to  be  inoculated,  while  the  sealed  end  is 
gradually  brought  into  a  flame.  The  vapor  thus  produced  will  drive 
out  the  bacterial  liquid. 


FIG.  62.  Sealing  of  cultures  in  capillaries,  a— Pipette  c  Fig.  61  drawn  out.  When 
filled  with  liquid  it  is  sealed  at  x.  b — The  method  of  removing  the  contents  from  a  capillary 
bulb  tube.  A  piece  of  rubber  tubing  x  slips  over  the  end  of  the  large  sterile  tube;  £— Fill- 
ing and  sealing  a  capillary  for  thermal  death-point  determination. 

The  removal  of  the  liquid  from  the  bulb  tube  can  be  accom- 
plished in  the  manner  indicated;  or,  by  means  of  the  arrangement 
shown  in  Fig.  62  b.  One  end  of  the  narrow  tube  is  opened  and  steril- 
ized by  flaming.  The  other  end  is  then  opened  and  inserted  into  a 
sterile  glass-tube,  the  end  of  which  is  provided  with  a  short  piece  of 
rubber  tubing.  By  gently  blowing  into  the  tube  the  contents  of  the 
capillary  can  be  expelled  into  a  test-tube  or  other  receiver. 

The  ordinary  chemical,  graduated  pipettes1  are  fre- 
quently made  use  of.  The  short  18  cm.,  1  c.c.  pipettes,  as 
used  in  water  analysis,  are  sterilized  in  a  sheet-iron  box 


1 A  set  of  three  pipettes  of  excellent  construction,  with  a_capa- 
pa 
Paris, 


ipa- 

>acity  of  TV,  rta  an(l  nrW  c.c.  respectively,  can  be  obtained  of  Ruelle, 
Rue  Chouin. 


460  BACTERIOLOGY. 

similar  to  that  used  for  sterilizing-  plates.  The  larger 
pipettes  must  be  protected  in  a  different  way  against  sub- 
sequent contamination  from  the  air.  The  simplest  pro- 
cedure is  to  push  a  short  cotton  plug  into  the  mouth  end  of 
the  pipette.  The  delivery  end  is  inserted  through  a  cotton 
plug  into  a  test-tube.  The  pipettes,  thus  protected,  are 
then  sterilized  in  the  usual  way. 

Drawing  of  Blood, 

In  studying  agglutination  and  in  many  other  experi- 
ments it  is  necessary  to  draw  a  small  quantity  of  blood  from 
an  animal.  When  it  is  desired  to  obtain  sterile 
blood-serum  the  blood  must  be  drawn  under 
strictly  aseptic  conditions.  It  should  be  re- 
ceived in  a  sterile  pipette  similar  to  that  shown 
in  Fig.  63. 

This  pipette,   which  is  a  slight  modification  of 
Nuttall's,  can  be  readily  prepared.     A  piece  of   glass 
tubing-  about  2.5  cm.  in  diameter  and  50  cm.  in  length 
is  selected.      A   medium   flame   from    a   blast-lamp  is 
directed    against    the    middle  of  the    tube.      As    the 
tube  is  slowly  rotated  and   heated   the   glass  softens 
and  a  narrow  constriction  results.     When  constricted 
to    about    one-third    the    original  diameter,  the  two 
halves  are  drawn  apart  so  that  the  parallel  tubes  are 
connected  at  an  angle  of  about  120°  by  the  resulting 
capillary.     They  are  finally  separated  by  sealing  the 
latter  at  the  mtddle  in  a  flame.     After  plugging  with 
cotton,  the  pipettes  are  sterilized  in  a  dry-heat  oven. 
The  capillary   tip    on    each    tube    should  not 
exceed  7  or  8  cm.     Moreover,  it  is  important 
that  the  capillary  should  not   be  too  narrow. 
FIG.  63.    Pipette  for  draw-  The  tip  should  be  about  1.5  to  2  mm.  in  di- 
ie?um°°*  t0  °btain  Stedle  ameter  in  order  to  obtain   the   best  yield  of 

,          blood; 

In  the  absence  of  the  wide  glass-tubing  indicated  above,  an  ex- 
cellent pipette  can  be   made  out  of   a  large  wide   test-tube.     The 


DRAWING  OF  BLOOD.  461 

bottom  of  this  afrd  of  a  small  test-tube  should  be  heated  in  the  blast- 
lamp  and  then  fused  together.  A  narrow  'or  medium  flame  should 
then  be  directed  against  the  large  test-tube  at  about  2  or  3  cm.  from 
the  end.  On  slow  rotation  and  careful  heating  a  thickened  constric- 
tion results.  The  two  portions  are  then  drawn  apart  and  the  capil- 
lary sealed  as  above. 

The  carotid  artery  of  the  anesthetized  animal  is 
exposed  and  a  sterile  silk  thread  is  slipped  under  the 
vessel.  A  pair  of  pression  forceps  are  applied  to  the  artery 
as  far  up  the  neck  as  possible;  or  the  artery  may  be  tied 
in  this  place.  A  second  pair  are  then  applied  about  3  cm. 
below  this  point.  The  distended  artery  is  grasped  with  an 
ordinary  wide-tipped  forceps,  just  below  the  upper  clamp, 
and  slightly  stretched.  It  is  then  nicked  with  a  pair  of  very 
fine  scissors.  After  inserting-  the  blades  of  a  narrow-pointed 
pair  of  forceps  the  vessel  can  be  distended  so  as  to  readily 
admit  the  introduction  of  the  open  end  of  a  pipette.  The 
end  of  the  pipette,  which  should  not  be  too  narrow,  is 
broken  off,  and  flamed  to  fuse  the  sharp  edges;  when  cool 
it  is  inserted  as  far  as  possible  into  the  vessel.  The  liga- 
ture is  then  tied  over  the  glass  tip;  or  this  may  be  held 
between  the  thumb  and  fore-finger.  When  the  lower  artery 
forceps  are  opened,  the  blood  rapidly  fills  the  sterile  pipette. 
If  necessary,  suction  can  be  applied  to  the  end  of  the  tube. 
When  enough  blood  has  been  drawn  the  artery  is  again 
clamped,  and  the  pipette  is  then  removed  and  sealed  in  a 
blast-lamp.  The  artery  should  then  be  tied  above  and 
below  the  wound.  After  the  blood-serum  separates  from 
the  clot  it  can  be  transferred  to  sterile  tubes  by  means  of 
a  bulb  pipette  (Fig.  61  e). 

When  only  a  small  quantity  of  blood  is  desired  it  can  be  obtained 
more  easily  from  the  jugular  vein.  Small  bulb  pipettes  (Fig.  61  e)  can 
be  employed  in  the  manner  indicated  above.  Moreover,  a  syringe 
can  be  used  to  advantage  in  drawing  blood  from  a  vein.  Several  c.c.. 
of  blood  can  thus  be  drawn  in  a  few  moments  from  the  external 
jugular  or  from  the  ear  vein  of  a  rabbit. 


462  BACTERIOLOGY. 

Small  quantities  of  human  blood  can  be  obtained  in  the 
following-  manner:  A  piece  of  rubber  tubing-  is  tied  above  the 
elbow  in  order  to  compress  the  surface  veins.  On  strongly 
flexing  the  arm,  the  superficial  veins  on  the  extensor  surface 
will  stand  out  prominently.  The  median  cephalic,  or 
basilic  veins  can  also  be  used.  The  surface  of  the  skin 
over  one  of  these  larg-e  veins  should  be  thoroughly  washed 
and  disinfected.  The  hypodermic  syringe  (2  c.c.),  provided 
with  a  wide  needle,  is  sterilized  by  boiling-  in  water  for  15 
minutes.  When  cold,  the  needle  is  inserted  into  the  vein 
without  the  slig-htest  difficulty.  The  piston  is  then  slowly 
withdrawn  and  the  syringe  fills  with  blood.  As  the  needle 
is  withdrawn  the  opening-  is  closed  with  the  finger  and 
eventually  an  antiseptic  compress  and  bandage  is  applied. 

Scarcely  any  pain  is  experienced  by  the  subject  and  the 
operation  is  borne  a  great  deal  better  than  if  a  lance  were 
used.  A  quantity  of  sterile  blood  can  thus  be  obtained  in  a 
iew  minutes. 

In  order  to  obtain  serum  for  testing  the  agglutinating 
power,  or  for  other  purposes  the  blood  is  at  once  forced  out 
into  a  centrifuge  tube.  It  should  be  thoroughly  whipped 
with  a  narrow  glass  rod  and  finally  centrifugated.  The 
•clear  serum  is  then  taken  up  in  a  pipette  and  tested. 

The  blood  of  the  horse  can  be  obtained  readily,  and  in 
large  quantity,  by  introducing  a  trochar  into  the  jugular 
vein.  This  method  of  bleeding  is  followed  in  the  prepara- 
tion of  antitoxin.  The  operation  is  carried  out  under  asep- 
tic conditions  and  hence  the  blood-serum,  thus  obtained,  is 
sterile.  The  method  of  procedure  is  essentially  that  given 
on  p.  268.  The  trochar  is  connected  by  means  of  a  sterile 
rubber  tube  with  a  short  glass  tube.  This  is  passed  into 
the  sterile  cylinder  or  battery  jar.  The  latter  should  be 
covered  with  a  double  layer  of  paper  before  sterilization. 
The  outer  paper  is  then  removed  and  the  glass  tube  is 
punched  through  the  inner  paper  cover.  From  4  to  6  liters 
of  blood  can  be  drawn  from  a  horse  at  one  bleeding. 


BLOOD-SERUM.  463 

Oxalate  blood  or  plasma. — As  is  well  known,  the  coagulation  of  blood 
can  be  prevented  by  the  addition  of  a  small  quantity  of  potassium 
oxalate.  The  author  has  utilized  this  fact  in  order  to  obtain  fluid 
blood  or  unaltered  plasma  for  culture  purposes. 

A  6  per  cent,  potassium  oxalate  solution  is  prepared.  The 
capacity  of  the  pipette  (Fig.  63)  is  ascertained  and  the  necessary 
amount  of  oxalate  solution  is  added  so  that  the  collected  blood  will 
have  0.1 — 0.2  per  cent,  of  potassium  oxalate.  Thus,  if  the  pipette 
can  hold  25  c.c.  of  blood,  then  0.8  c.c.  of  the  oxalate  solution  should 
be  added  in  order  that  the  resulting  mixture  shall  contain  0.2  per 
cent,  of  oxalate.  The  tube  and  contents  are  then  sterilized  in  the 
autoclave. 

Before  inserting  the  tip  of  the  pipette  into  the  artery,  the 
oxalate  solution  should  be  rolled  so  as  to  moisten  the  inner  wall  of 
the  pipette.  This  is  then  filled  with  blood  and  sealed  according  to 
the  directions  given  above. 

The  pipettes  can  be  placed  in  an  ice-chest,  in  which  case  the 
corpuscles  will  subside  in  2  or  3  days,  and,  as  a  result,  a  perfectly  clear 
plasma  can  be  obtained.  This  may  be  transferred  by  means  of  a  bulb 
pipette  to  sterile  tubes.  It  can  be  used  as  such,  or  can  be  solidified 
as  in  the  case  of  blood-serum. 

Similarly,  the  fluid  blood  itself  may  be  placed  in  tubes  and  c'oagu- 
lated  at  about  70°.  The  addition  of  1  part  of  oxalate  blood  to  2  parts 
of  melted  agar  at  50°  yields  a  bright  red  medium  which  can  be  solidi- 
fied in  an  inclined  position.  This  blood-agar  is  an  excellent  medium 
for  streptococci  and  other  organisms. 


Blood-Serum. 

The  serum  from  the  blood  of  the  ox  is  frequently  em- 
ploy ed%  for  cultivation  purposes.  The  blood  as  it  is  ordin- 
arily collected  in  the  slaughter  house  is  not  sterile.  If, 
however,  it  is  received  into  sterile  vessels  and  kept  covered 
at  a  low  temperature  the  few  organisms  that  are  present 
will  not  multiply,  and  hence,  a  large  proportion  of  the  tubes 
filled  with  such  serum  will  remain  sterile. 

The  blood  should  be  received  in  battery  jars  which  have 
been  covered  with  paper  and  sterilized.  When  the  blood 
has  formed  a  solid  clot,  it  is  transferred  to  the  ice-chest. 


464  BACTERIOLOGY. 

A  considerable  amount  of  serum  separates  in  from  24-48 
hours.  It  should  be  drawn  off  by  means  of  a  volume 
pipette  and,  if  not  perfectly  clear,  it  should  be  set  aside 
again  for  a  day  to  allow  the  corpuscles  to  settle.  The 
serum  thus  obtained  can  be  sterilized  by  one  of  the  follow- 
ing methods. 

Sterilization  of  serum  by  filtration. — Clear,  thoroughly 
sedimented  blood-serum  is  very  desirable  in  order  to  obtain 
rapid  nitration.  The  serum  is  filtered  through  an  unglazed,. 
porcelain  Pasteur-Chamberland,  or  through  a  Berkefeld  in- 
fusorial earth  bougie.  The  Pasteur  filters  differ  greatly  in 
their  flow  and  this  is  due  chiefly  to  the  variable  thickness 
of  the  wall.  In  most  bougies  the  wall  has  a  thickness  of 
2.5  to  2.8  mm.  and  through  such,  blood-serum  can  be  filtered 
with  great  difficulty.  Occasionally  a  bougie  is  met  with  in 
which  the  wall  is  less  than  2  mm.  thick,  and  through  these 
the  blood-serum  can  be  filtered  with  ease. 

The  Berkefeld  filter  is  considerably  more  porous  than  that  of 
Pasteur  and  hence  can  be  used  to  advantage  in  the  filtration  of 
blood-serum.  A  pressure  of  at  least  75  Ibs.  to  the  square  inch  should 
be  employed.  Obviously,  the  less  porous  the  filter  the  higiier  the 
pressure  that  must  be  employed.  When  a  liquid  contains  protein 
substances  it  should  always  be  filtered  under  as  hig-h  a  pressure  as 
possible. 

The  filtering-  apparatus  (Fig-.  66,  p.  469),  permits  the  use  of 
either  the  Pasteur  or  the  Berkefeld  filter,  and  can  be  used  with  or 
without  pressure.  The  -filters  are  sterilized  according-  to  the  direc- 
tions given.  The  filtered  serum  may  be  received  in  a  sterile  Erlen- 
meyer  vacuum  flask;  or,  in  a  globe  receiver  such  as  is  shown  in  Fig, 
68  B.  In  the  latter  case  the  filtrate  can  be  transferred  to  tubes  or 
flasks,  with  a  minimum  risk  of  contamination. 

Fractional  sterilization  of  serum  at  58°. — Blood-serum 
coagulates  at  about  70°  to  an  opaque,  white  mass.  When, 
therefore,  it  is  desired  to  obtain  a  sterile,  liquid  serum,  or  a 
solid,  transparent  serum  it  is  necessary  to  resort,  either  to 
the  procedure  just  given,  or  to  fractional  sterilization,  at  a 


BLOOD-SERUM.  465 

lower  temperature  than  that  mentioned.  A  temperature  of 
58°  maintained  for  an  hour  will  destroy,  as  a  rule,  actively 
growing  bacteria.  The  spores,  of  course,  are  not  affected 
by  this  heat.  They  must  be  given  an  opportunity  to  ger- 
minate, in  which  case  the  resistant  spore  is  converted  into 
a  relatively  weak,  vegetating  form.  When  this  takes  place 
the  latter  promptly  yields  to  the  temperature  employed. 
It  should  be  remembered,  however,  that  there  are  bacteria 
which,  far  from  being  killed,  actually  thrive  at  this  tem- 
perature'(p.  72).  If  these  should  happen  to  be  present  it 
will  be  impossible  to  sterilize  the  serum  by  this  method. 
The  sterilization  of  the  serum  tubes  can  best  be  accom- 
plished by  the  "use  of  a  Roux  water-bath.  This  exceedingly 
useful  apparatus  is  shown  in  Fig.  64,  and  as  shown,  it  is 
provided  with  a  Roux  metallic  thermo-regulator  (R).  A 
wire  basket  (D)  is  immersed  in  the  water  and  is  provided 
with  an  adjustable  bottom  (E).  In  this  way  the  tubes  can 
be  immersed  in  water  to  any  desirable  depth.  The  cover 
(C)  is  double- walled  and  filled  with  water.  Through  the 
opening  on  the  top  a  thermometer  is  inserted  into  the  liquid. 

The  wire  basket,  as  ordinarily  supplied  with  this  apparatus,  is 
provided  with  a  movable  bottom  which  is  clamped  to  the  central 
tube.  The  latter  takes  up  desirable  space  and  it  is  advisable,  there- 
fore, to  alter  the  basket  so  that  the  bottom  can  be  clamped,  on  the 
under  side,  to  runners  fastened  on  the  inner  surface  of  the  wire 
basket.  The  thermometer  and  mercury  thermo-regulator  (Fig-.  37, 
p.  246),  can  be  suspended  to  advantage  in  the  side  compartment  which 
is  intended  for  the  metallic  regulator. 

The  serum  tubes  are  sterilized  in  this  apparatus  by 
heating  at  58°  for  one  hour  on  each  of  six  consecutive  days. 
The  interval  between  heating  may  be  shortened  to  10  hours 
if  the  tubes,  after  each  heating,  are  placed  for  2-3  hours  at 
37°.  This  will  assist  the  germination  of  the  spores  present. 
The  tubes  are  then  kept  at  the  ordinary  room  temperature 
for  about  eight  hours,  after  which  they  are  submitted  again 
to  a  heat  of  58°. 

30 


BACTERIOLOGY. 


A  temperature  of  58°,  maintained  continuously  for  6-8 
hours,  will  cause  the  coagulation  of  blood-serum.  Loffler's 
serum  and  glycerin  serum  are  not  as  readily  affected. 

Fractional  sterilization  of  serum  at  75-80°. — At  this  tem- 
perature ordinary  blood- serum,  and  even  Loffler's  serum  will 

coagulate.  Conse- 
quently, only  solidified 
serum  will  be  obtained 
by  this  procedure.  The 
method  takes  less  time 
than  the  preceding.  It 
yields  an  almost  per- 
fectly transparent  yel- 
low medium  which  is 
a  decided  advantage 
over  the  method  to  be 
described  next.  The 
cotton  plugs  of  the 
serum  tubes  should  be 
cut  off  close  to  the 
end  of  the  tube  which 
should  then  be  turned 
in  a  flame  till  the  cot- 
ton begins  to  change 

FlG.  64.     The  Roux  water-bath  for  serum  sterilization. 

D— Wire  basket  with  adjustable  bottom  E;  R— Metallic  Color.  The  tubes  are 
regulator  of  Roux. 

then    sealed    with 

sterile,  rubber  caps.  This  precaution  insures  the  steriliza- 
tion of  the  cotton  plug,  and  hence,  prevents  subsequent 
contamination  by  moulds.  Moreover,  the  serum  when  once 
solidified  will  not  dry  down  during  the  subsequent  exposures 
to  heat. 

The  serum  tubes,  sealed  as  above,  are  inclined  in  what  is  known 
as  Koch's  serum  sterilizer  (Fig-.  65).  The  inner  compartment  is  sur- 
rounded by  a  water-jacket.  A  thermometer  should  be  placed  on  the 
inside  of  the  apparatus  and  another  one  is  suspended  in  the  water.  A 


BLOOD-SERUM. 


467 


thermo-reg-ulator  is  employed  to  secure  a  constant  temperature. 
Heat  is  applied  till  a  temperature  of  about  75°  is  reached  and  this  is 
then  maintained  for  1  hour.  This  is  repeated  on  each  of  three  or  four 
successive  days.  The  time  necessary  to  secure  sterilization  can  be 
shortened  by  transferring-  the  tubes,»for  a  few  hours  after  each  heat, 
to  an  incubator  at  37°  as  described  in  connection  with  the  preceding- 
method. 

Sterilization  of  serum  at  100° . — Serum  sterilized  at  this 
temperature  is  opaque  white.  This,  however,  does  not  in- 
terfere with  its  value  as 
a  nutrient  medium.  The 
loss  of  transparency  is 
counterbalanced  by  the 
ease  with  which  it  can 
be  prepared  in  larg-e 
quantities.  If  inclined 
blood-serum  is  heated 
rapidly  to  100°  it  will 
solidify,  but  the  mass 
becomes  torn  up  by  gas 
bubbles  due  to  the  expul- 
sion of  carbonic  acid. 

The  formation  of 
these  bubbles  or  spaces  can  be  avoided  if  the  serum  is  first 
maintained  at  a  temperature  of  about  80°  for  some  time. 
For  this  purpose,  they  can  be  inclined  in  an  ordinary  air- 
bath  (Fig-.  25),  or  in  the  apparatus  shown  in  Fig*.  65.  After 
keeping-  the  serum  at  80°  for  some  time,  the  temperature 
should  be  gradually  raised  till  that  of  100'  is  reached.  The 
tubes  are  then  steamed  for  30  minutes  on  each  of  3  suc- 
cessive days. 

Solidification  of  serum. — The  sterile,  fluid  serum  can  be 
used  as  such,  but  more  often  it  is  coagulated  in  an  inclined 
position  and  employed  for  streak  cultures.  The  fluid  serum 
should  be  kept  in  sealed  tubes  to  prevent  evaporation  (see 


FIG.  65.    Koch's  serum  sterilizer. 


468  BACTERIOLOGY. 

Chapter  XV).  When  inclined  serum  is  wanted  the  tubes  are 
placed  in  the  coagulating-  apparatus  (Fig-.  65),  and  heated 
for  about  1  hour  at  70°.  They  are  then  ready  for  use. 
Lsffler's  blood-serum  and  glycerin  serum  must  be  heated  to 
a'bout  75°  in  order  to  insure  perfect  coagulation.  Obviously, 
the  blood-serum  tubes  may  be  inclined  in  the  ordinary  dry- 
heat  sterilizer  (Fig.  25)  and  solidified  at  70°,  or  at  a  higher 
temperature. 

Ldffler's  blood- serum. — This  medium  is  chiefly  employed 
for  cultivating  the  diphtheria  bacillus.  It  is  prepared  by 
adding  to  three  parts  of  blood-serum,  one  part  of  ordinary 
bouillon  to  which  1  per  cent,  of  glucose  has  been  added. 
The  mixture  is  then  sterilized  according  to  one  of  the  meth- 
ods outlined  above.  It  is  finally  coagulated  in  an  inclined 
position.  It  should  be  remembered  that  L<3ffler's  serum  re- 
quires more  time  and  a  higher  temperature  to  secure  coagu- 
lation than  does  ordinary  serum. 

Glycerin  blood-serum. — This  is  prepared  by  merely  adding-  5-6  per 
cent,  of  glycerin  to  the  blood-serum.  This  mixture  is  then  sterilized 
and  inclined  according-  to  the  directions  given  above. 

Boiled  non-coagulated  serum. — When  serum  is  diluted  with  5  to  10 
parts  of  distilled  water  it  can  be  sterilized  by  steaming  without 
undergoing-  coag-ulation.  This  albuminous  liquid,  can  be  utilized  to 
advantage  in  some  cases. 

Oxalate  blood  or  plasma. — The  preparation  of  this  medium  is  de- 
scribed on  p.  463. 


Filtration  of  Bacterial  Liquids. 

A  liquid  may  be  deprived  of  the  bacteria  which  are 
present  by  filtration  through  unglazed  porcelain.  The 
Pasteur-Chamberland  bougie  is  by  far  the  most  reliable  for 
this  purpose.  As  stated  on  p.  464  the  walls  of  the  bougies 


FILTRATION  OF    BACTERIAL,  LIQUIDS. 


469 


vary  somewhat  in  thickness,  and  hence  the  rate  of  flow  is 
variable.  Highly  albuminous  fluids  cannot  be  filtered  un- 
less the  wall  of  the  filter  is  relatively  thin  or  unless  ~ex^ 
tremely  high  pressure  is  employed  to  force  the  soluble  pro- 
teins through  the  pores  of  the  filter.  With  insufficient 
pressure  these  remain  on  the  filter  and 
only  an  aqueous  liquid  will  pass  through. 

The  liquid  to  be  filtered  should  con- 
tain as  little  suspended  matter  as  possible. 
For  this  reason,  blood-serum  should  be 
centrifugated  or  allowed  to  settle  until 
free  from  corpuscles.  Bouillon  cultures 
should  first  be  filtered  through  several 
thicknesses  of  paper.  The  hardened, 
parchment-like  paper  (No.  575)  of  Schlei- 
cher  and  Schiill  is  well  adapted  for  remov- 
ing the  mass  of  bacteria  from  a  liquid. 

A  larg-e  number 
of  filters  have  been 
devised,  but  most  of 
these  are  far  from 
being  satisfactory 
for  ordinary  labora- 
tory work.  One  of 
the  best  filters  is  that  of  Martin,  but  it  can  be  used  only 
with  negative  pressure, — that  is  to  say  by  producing  a 
vacuum  in  the  receiving  flask.  The  author  has  described 
a  filter1  which  has  been  found  to  be  very  useful  for  labor- 
atory work.  It  can  be  connected  with  an  air  pump,  or 
additional  pressure  may  be  obtained  by  connecting  with  a 
cylinder  of  compressed  air.  It  can  be  used  for  filtering 
large  or  small  quantities  of  liquid. 

The  apparatus  shown  in  Fig-.  66  consists  essentially  of  a  glass 
cylinder  (g)  3  cm.  in  diameter  and  20  cm.  in  length.     The  upper  end  is 

1  Centralblatt  fur  Bakteriologie  22,  p.  337,  1897. 


FIG.  66.   Apparatus  for  filtering  bacterial  liquids  (F.  G.  N.). 


470 


BACTERIOLOGY. 


provided  with  a  globe  or  reservoir  having  a  capacity  of  250,  500  or  1000 
c.c.  The  lower  end  is  provided  with  a  flange  (/)  which  is  2  cm.  wide 
and  about  yz  cm.  thick.  The  flange  is  .ground  on  the  lower  surface. 
The  upper  surface  of  the  flange,  in  the  position  shown  in  Fig.  66, 
should  be  parallel  with  the  ground  surface  and  not  sloping.  This  is 
necessary  in  order  to  prevent  the  clamps  from  slipping  off.  The 
ground  surface  of  the  flange  must  form  a  perfect  right  angle  with 
the  inner  wall  of  the  cylinder  (see  Fig.  67).  This  is  necessary  in  order 
to  give  a  proper  support  to  the  shoulder  of  the  bougie. 


67. 


The  manner  of  obtaining  a  perfectly  tight  joint  is  shown  in  Fig. 
Before  proceeding  to  make  the  connections  the  cylinder  should 

be  inverted  so  that  the  globe  rests  on 
a  small  ring  of  a  retort  stand,  with  the 
flange  uppermost.    A  rubber  ring  (Fig. 
4-  67,  1)  is  slipped  over  the  bougie  and 
brought  up  against  the  shoulder.    This 
s  ring  should  not  be  made  of  very  soft 
rubber  inasmuch  as  it  would   expand 
laterally  and  crush  the  bougie  when 
the    clamps    are    applied.      Ordinary 
cloth-covered  rubber,  from  which  the 
cloth  has  been  torn  will  answer  the 
purpose  very  well.     The  rubber  ring 
,-Flanle  o^c^fef  /^Sho^iS'of  is  2-3  mm.  thick  and  5  cm.  in  diameter. 

filter;  .  i,  2  and  3-Rubber  rings;  4-Metal    The  circular  opening  should  be   CUt  SO 

as  to  allow  the  ring  to  slip  easily  over 

the  bougie  (about  2.7  cm.  in  diameter).  The  bougie  provided  with  the 
rubber  ring  is  then  inserted  into  the  cylinder. 

Another  rubber  ring  (Fig.  67,  2)  is  now  slipped  over  the  mouth  of 
the  bougie.  This  ring  is  l>^-2  mm.  thick,  4  cm.  in  diameter  and  has  a 
central  opening  which  is  1.3cm.  in  diameter.  The  thick  rubber  ring 
(Fig.  67,  3)  is  then  placed  in  position.  This  should  be  14-15  mm.  thick 
and  should  be  7  cm.  in  diameter.  The  central  opening  is  cut  slanting 
so  that  the  upper  diameter  is  ±y2  cm.,  while  that  below  is  5>^  cm.  A 
brass  or  iron  plate  about  2  mm.  in  thickness  is  then  placed  on  top. 
This  plate  has  the  same  diameter  (7  cm.)  as  the  flange,  and  the  central 
opening  is  2.2  cm.  in  diameter. 

Three  clamps  such  as  are  employed  in  connection  with  the 
author's  anaerobic  apparatus  (p.  313),  are  then  applied.  These  are 
sufficient  for  vacuum  filtration,  but  in  case  the  liquid  is  to  be  sub- 
jected to  a  positive  pressure  of  60  or  80  pounds,  it  is  well  to  add  a 
fourth  clamp. 


FILTRATION   OF    BACTERIAL  LIQUIDS. 


471 


The  mouth  of  the  sterile  bougie  is  now  flamed  and  connected 
with  the  sterile  glass  tube  which  passes  through  the  rubber  stopper. 
A  glass  globe  similar  to  that  of  Martin  can  be  used  in  place  of  an 
Erlenmeyer  flask  to  receive  the  filtrate  (Fig.  68  B).  The  cylinder  is 
now  inverted  and  connected  with  the  receiver  (Fig.  66  e). 

Ordinary  liquids  can  be  filtered  by  the  aid  of  a  Chapman  aspira- 
tor. If  the  liquid  under  these  conditions  filters  slowly,  the  neck  of 
the  globe  (Fig.  66  h)  should  be  connected 
with  a  tank  containing  compressed  air. 
When  pressure  is  applied  care  must  be 
taken  to  prevent  the  stopper  or  the  glass 
tube  from  being  blown  -out  of  place. 
The  end  of  the  glass  tube  after  it  has 
been  slipped  through  the  rubber  stopper 
is  softened  in  the  flame  and  then  flanged 
by  means  of  a  piece  of  charcoal  or  a  nail. 
The  stopper,  when  inserted,  should  be 
wired  securely  in  place.  A  brass  ring 
(ft),  placed  on  the  upper  side  of  the  stop- 
per, will  prevent  it  from  being  cut  by 
the  wires. 

The  compressed  air  is  contained  in 
a  small  steel  cylinder  such  as  is  used  by 
physicians  for  atomizing  purposes.  A 
cheap  substitute  can  be  made  by  con- 
necting a  domestic,  hot-water  tank  with 
a  pressure  gauge  and  a  bicycle  pump. 


The  Berkef  eld  filter  can  be  attached  FIG  58  A  Berkefeld  filter  showing 
to  the  glass  cylinder  in  a  somewhat  simi-  yS^SSfSf^^.^Kf. 
lar  manner.  An  iron  plate  (Fig.  68  6)  5-6 

mm.  thick  and  7  cm.  in  diameter  is  provided  with  a  central  opening 
(1.2  cm.  diameter)  just  sufficient  to  allow  the  nozzle  of  the  filter  to  pass 
through.  A  rubber  ring  (Fig.  68  a),  7  cm.  in  diameter  and  2  mm.  thick, 
with  a  central  opening  3  cm.  in  diameter,  is  placed  between  the  glass 
flange  and  the  iron  plate.  The  whole  is  then  clamped  securely.  . 

In  place  of  the  Erlenmeyer  vacuum  flask  (Fig.  66  e),  a  glass  globe 
like  that  shown  in  Fig.  68  B  can  be  employed  to  advantage.  The  three 
tubes  (d,  e,  /)  are  plugged  with  cotton  and  the  globe  is  then  sterilized 
in  the  dry-heat  oven.  The  tube  d,  after  removal  of  the  plug,  is  con- 
nected with  the  bougie  by  means  of  the  sterile  rubber  tube  c.  The 
tube  /  is  connected  in  like  manner  with  a  rubber  tube  to  the  drawn- 


472  BACTERIOLOGY. 

out  glass  tube  g.  The  receiver  and  bougie  are  now  heated  in  an  auto- 
clave to  insure  sterilization.  Finally,  the  tube  e,  with  the  cotton  plug- 
in  place,  is  connected  with  the  pump  in  the  manner  indicated  in  Fig-. 
66.  After  filtration,  the  tube  d  is  disconnected  and  plugged  with 
sterile  cotton  taken  from  an  ordinary  sterile  tube.  The  tube  g  is 
drawn  out  to  a  narrow  capillary  and  sealed  at  the  end.  In  order  td 
remove  the  liquid  from  the  receiver,  the  end  of  the  tube  g  is  broken, 
flamed  and  inserted  into  the  flask  or  tube  to  be  filled. 

The  bougies,  connecting-  tubes  and  receiving"  flasks  must  be  per- 
fectly sterile.  The  porcelain  bougies  should  be  scoured  with  sand- 
paper, dried  and  then  sterilized  either  by  the  direct  heat  of  a  flame, 
or  in  a  dry  heat  sterilizer.  It  is  better  to  sterilize  the  moist  bougie 
rh  an  autoclave  at  120°  for  half  an  hour.  The  mouth  of  the  bougie 
should  be  plugged  with  cotton  and  the  entire  bougie  should  be  wrap- 
ped in  paper.  The  Berkefeld  filters  are  sterilized  by  boiling-  in  water 
or  by  steaming-  in  an  autoclave.  After  filtration,  the  entire  apparatus 
should  be  sterilized  by  steam. 

The  receiving-  flask  (Fig-.  66  e)  is  connected  with  a  tube  (d)  filled 
with  sterile  sand.  This  serves  to  prevent  bacteria  from  entering-  the 
flask  when  air  is  admitted  through  the  glass  stopcock  c.  The  flask  b 
serves  to  collect  any  back-flow  that  may  come  from  the  aspirator. 


Tuberculin. 

Tuberculin  contains  essentially  the  soluble  products  of 
the  tubercle  bacillus.  The  organism  growing  in  a  suitable 
medium  gives  rise  to  chemical  products,  some  of  which  pass 
out  into  the  surrounding  liquid.  The  bacterial  cells  are  re- 
moved by  filtration  and  the  clear  liquid,  after  concentration, 
is  known  as  tuberculin.  It  can  be  prepared  according  to 
the  following  directions : 

Ordinary  bouillon  is  prepared  from  beef  or  veal,  and  5 
per  cent,  of  glycerin  is  added.  This  medium  is  then  filled 
into  small  Erlenmeyer  flasks  to  a  depth  of  about  1  inch. 
A  broad,  low  flask  provided  with  a  loosely  fitting,  glass  cap 
is  very  useful.  The  flasks  should  be  provided  with  very 
firm  cotton  plugs.  The  flasks  of  bouillon  are  sterilized  in 
steam  in  the  usual  way. 


TUBERCULIN.  473 

They  are  then  inoculated  with  the  tubercle  bacillus  in 
such  a  manner  that  the  material  planted  will  remain  float- 
ing- on  the  surface  of  the  liquid.  This  can  be  done  by  means 
of  a  looped  platinum  wire  which  should  be  bent  at  right 
angles,  a  short  distance  from  the  loop.  A  large  piece  of 
the  dryish  growth  is  loosely  picked  up  on  the  loop  which, 
as  it  is  passed  into  the  liquid,  leaves  the  mass  behind  on 
the  surface.  A  thin  piece  of  cork  about  1  cm.  square  may 
be  placed  in  the  bouillon  and,  after  sterilization,  the  tuber- 
cle bacillus  may  be  transferred  to  this  by  means  of  a  Roux 
spatula  (Fig.  49  a,  p.  278).  The  cotton  plugs  of  the  flasks 
are  then  charred  and  covered  with  caps  of  filter-paper.  The 
flasks  are  carefully  placed  in  an  incubator  at  39°  and  they 
remain  there  for  4  or  5  weeks  or  more.  The  first  indica- 
tions of  a  growth  will  be  seen  in  about  two  weeks.  After 
that  the  growth  is  more  rapid,  and,  about  the  third  week,  a 
thin  folded,  whitish  scum  will  cover  the  entire  surface.  A 
yeast-like  odor  will  pervade  the  incubator.  Eventually,  the 
growth  which  spreads  over  the  surface  forms  a  thick,  folded, 
yellowish,  dry  mass. 

The  flasks  are  then  steamed  for  1  hour,  after  which  the 
contents  are  filtered  through  paper  (p.  469).  The  yellowish 
liquid  may  then  be  concentrated  on  the  water-bath  to  about 
rVth  the  original  volume,  or  it  may  be  filtered  at  once 
through  a  Pasteur  or  Berkefeld  filter  and  placed  in  sterile 
bottles.  One  half  per  cent,  of  carbolic  acid  may  be  added 
as  a  preservative. 

A  more  convenient  procedure  is  to  cultivate  the  tubercle  bacil- 
lus on  glycerin  potatoes  in  large  Roux  tubes.  These  should  not  be 
tig-htly  sealed  with  rubber  caps  or  with  wax,  but  should  be  closed  with 
a  cork  stopper  which  is  provided  with  a  drawn-out  glass  tube  (Fig-.  54, 
p.  315).  The  lower  portion  of  the  potato  should  be  in  contact  with  5 
per  cent,  aqueous  glycerin.  Every  two  or  three  days  the  liquid  should  be 
agitated  so  as  to  moisten  the  potato.  A  very  rich  growth  is  thus  ob- 
tained which  can  then  be  stirred  into  the  dilute  glycerin  and  finally  re- 
moved by  means  of  a  drawn-out  bulb  pipette  (Fig-.  61  e,  p.  457).  The  bac- 
terial suspension  is  then  steamed,  filtered  and  concentrated  as  above. 


474  BACTERIOLOGY. 

The  tuberculin  thus  prepared  should  be  tested  upon 
tuberculous  guinea-pigs.  The  fatal  dose  should  be  ascer- 
tained and  the  effect  on  the  temperature  of  the  animals 
should  be  observed. 


Diphtheria  Toxin. 

« 

The  diphtheria  bacillus  when  it  grows  in  bouillon  pro- 
duces a  powerful  toxin.  The  ordinary  bouillon  which  is  al- 
kaline at  the  time  of  inoculation  becomes  acid  in  about  24 
hours  and  then  gradually,  in  about  4  or  5  days,  it  returns  to- 
an  alkaline  condition.  The  toxin  is  produced  especially 
during-  the  alkaline  stage.  By  passing1  a  current  of  air 
over  the  liquid,  the  acid  stage  can  be  shortened  and,  hence, 
the  toxicity  of  the  culture  is  increased.  If,  however,  the 
aeration  is  continued  for  more  than  5  days  it  will  cause  an 
oxidation  of  the  toxin,  and,  as  a  result,  the  liquid  decreases 
in  toxicity. 

Bouillon  made  out  of  commercial  meat  extract  does  not 
give  rise  to  acid  products,  and  hence  such  media  have  been 
used  for  preparing*  toxins.  The  commercial  peptons  vary  a 
great  deal  in  their  composition,  and  while  some  give  a 
bouillon  which  does  not  change  in  reaction,  others  will  give 
rise  to  acid  products.  The  production  of  a  temporary  acid 
reaction  in  bouillon  made  out  of  meat  is  undoubtedly  due  to 
the  presence  of/sugar.  This  can  be  removed  by  allowing 
the  meat  to  ferment. 

The  bouillon  best  adapted  for  the  cultivation  of  the 
diphtheria  bacillus  is  prepared  as  follows: 

1.— 500  g.  of  chopped  beef  are  added  to  1000  c.c.  of  water;  the 
mass  is  thoroughly  mixed  and  set  aside  for  20  hours  at  35°.  The  diges- 
tion at  this  temperature  serves  to  destroy  the  sugar  that  may  be  pres- 
ent. The  liquid  is  then  strained  through  well  washed  muslin.  5  g.  of 
common  salt  and  20  g.  of  Witte's  pepton  (2  per  cent.)  are  added  to  1 
liter  of  the  filtrate.  The  liquid  is  then  titrated  with  TN¥  NaOH  in  the 


DIPHTHERIA   TOXIN.  475- 

manner  described  on  p.  155.  To  the  amount  of  normal  alkali  neces- 
sary to  neutralize  the  liquid  an  excess  of  alkali,  corresponding-  to  7  c.c. 
of  N  NaOH  per  liter,  is  added.  The  mixture  is  then  heated  to  70°  and- 
filtered  through  paper,  and  finally  through  porcelain.  The  sterile  fil- 
trate is  then  transferred  to  sterile  wide  flasks,  and  inoculated  with 
the  culture. 

The  bouillon  may  be  boiled,  filtered  and  then  sterilized  by  steam- 
ing on  each  of  3  successive  days,  or  by  heating-  in  the  autoclave  at 
110°.  Heating-,  however,  tends  to  alter  the  reaction  of  the  medium, 
and  hence  diminishes  the  toxicity  of  the  liquid.  Nevertheless,  steril- 
ization by  heat  is  more  convenient  and  hence  is  resorted  to  most 
often. 

2. — Instead  of  adding-  commercial  pepton  which,  as  indicated 
above,  is  liable  to  vary  in  composition,  Martin  adds  an  equal  volume 
of  a  pepton  solution  obtained  by  digesting-  the  stomach  of  a  pig-. 

This  is  prepared  as  follows:  A  clean  pig's  stomach  is  freed  from 
fat,  cut  up  into  small  pieces  which  are  then  passed  through  an  Enter- 
prise sausage  machine.  To  200  g.  of  the  finely  divided  tissue,  1  liter 
of  water  and  10  c.c.  of  concentrated  HC1  are  added,  and  the  mixture 
is  set  aside  at  50°  for  about  12  hours. 

The  digested  liquid  is  then  carefully  decanted  to  a  filter  of  ab- 
sorbent cotton  (Fig1.  35,  p.  237).  Inasmuch  as  the  filtrate  is  intensely^ 
acid  it  is  advisable  to  neutralize  the  excess  of  acid  by  the  addition  of 
a  strong  alkali.  This  can  be  done  by  adding  25  c.c.  of  a  16  per  cent.. 
NaOH  solution.  The  liquid  is  then  thoroughly  mixed  and  the  residual 
acidity  is  determined  by  titration  (see  p.  155).  To  the  amount  of 
N  alkali  necessary  to  neutralize  the  liquid,  an  excess  of  7  c.c.  per  liter 
should  be  added  in  order  to  impart  the  most  favorable  alkalinity. 

The  mixture  is  then  boiled  or  heated  at  120°  for  10  minutes. 
When  the  precipitate  settles,  the  liquid  can  be  filtered  through 
paper.  The  filtrate  will  be  perfectly  clear,  if  the  neutralization  was 
done  in  the  cold.  It  may  be  used  at  once,  or  it  may  be  placed  in 
flasks  and  sterilized  by  steam;  or  at  115°  for  15  minutes. 

In  the  pepton  solution  thus  prepared,  the  diphtheria  bacillus 
grows  without  producing  any  acidity.  To  make  the  culture  medium 
proper,  Martin  adds  to  this  pepton  solution  an  equal  volume  of  the 
fermented  meat  extract  (p.  474)  to  which  salt,  but  no  Witte's  pepton,. 
has  been  added.  The  mixture  is  then  titrated  and  given  the  alkalin- 
ity mentioned  above.  It  is  then  heated,  filtered,  filled  into  flasks  and 
sterilized  by  filtration  through  porcelain  or  by  steam.  This  medium 
as  stated  does  not  yield  acid  products,  and  it  can  give  rise  to  a  toxin, 
such  that  0.002  c.c.  (^  c.c.)  will  kill  a  500  g.  guinea-pig. 


476  BACTERIOLOGY. 

The  bouillon  prepared  as  above  under  (1),  or  the  mixed 
medium  described  under  (2)  is  inoculated  with  the  diph- 
theria bacillus  and  set  aside  at  37°.  A  scum  or  pellicle 
forms  on  the  surface  within  24  hours,  and,  if  this  is  shaken 
down,  another  one  will  form.  The  absence  of  a  pellicle  in- 
dicates a  weak  culture  or  an  unfavorable  medium.  The 
maximum  toxicity  is  reached  on  about  the  5th  to  the  7th 
day.  The  culture  should  then  be  filtered,  first  through 
paper,  then  through  porcelain.  This  should  be  done  under 
pressure  (p.  469).  After  the  10th  day  the  toxin  gradually 
diminishes.  The  filtered  toxin  can  be  transferred  to  sterile 
bottles,  preferably  of  dark-brown  glass;  or,  it  may  be 
drawn  up  into  sterile  bulb  pipettes  (p.  457)  which  are  then 
sealed  in  the  flame.  Inasmuch  as  air  tends  to  alter  the 
toxin  it  is  advisable  to  fill  the  bottle  or  pipette.  More- 
over, the  toxin  should  be  kept  in  a  cool,  dark  place.  A 
half  per  cent,  of  carbolic  acid  or  of  toluol  can  be  added  as 
.a  preservative.  The  strength  of  the  toxin  must  now  be 
ascertained  by  inoculating  a  series  of  animals. 

Determination  of  the  minimum  fatal  dose. — For  this  pur- 
pose a  number  of  guinea-pigs  are  selected  having  about  the 
same  weight  (250  g.).  Four  of  these  animals  should  be  in- 
jected subcutaneously  with  iV,  ^V,  TT><),  and  ^  c.c.  of  the 
filtered  toxin,  respectively.  The  small  quantities  are  meas- 
ured as  indicated  on  p.  479.  The  animal  may  die  in  a  few 
hours  or  not  until  after  the  lapse  of  two  or  three  weeks. 
In  the  latter  case,  post-diphtheritic  paralysis  of  the  ex- 
tremities is  likely  to  be  observed  for  some  days  previous  to 
•death.  The  animal  may  be  considered  as  having  recov- 
ered, when  it  increases  in  weight  and  has  a  normal  tem- 
perature. 

The  first  series  of  guinea-pigs  serves  to  establish  ap- 
proximately the  fatal  dose.  Thus,  if  the  guinea-pig  that 
received  ^V  c.c.  of  toxin  died  whereas  the  one  that  received 
rta  c  c.  survived,  it  is  evident  that  the  minimum  fatal  dose, 


DIPHTHERIA  TOXIN.  477 

which  is  the  amount  just  sufficient  to  kill,  lies  between 
these  two  limits.  Another  series  of  guinea-pigs  is  then 
taken  and  injected  with  ire,  A,  *V  and  TV  c.c.  respectively  odL 
the  filtered  toxin.  If  fa  of  a  c.c.  still  kills,  whereas  -fa  of  a 
c.c.,  although  it  sickens  the  animal,  does  not  kill,  it  fol- 
lows that  the  former  is  to  be  considered  as  the  minimum 
fatal  dose.. 

The  minimum  fatal  dose  of  a  filtered  diphtheria  culture 
will  vary  with  the  duration  of  culture,  the  medium  used, 
the  temperature  of  incubation  and  the  virulence  of  the  ba- 
cillus employed.  It  has  been  obtained  as  low  as  0.001  c.c., 
but  as  a  rule  it  is  above  0.01  c.c. 

In  accurate  work  it  is  desirable  to  inoculate  three  or  six  guinea- 
pigs  with  the  established  minimum  fatal  dose.  The  individual  ani- 
mals vary  more  or  less  in  resistance  and  it  is,  therefore,  necessary  ta 
make  certain  that  the  minimum  dose  is  surely  fatal,  not  merely  to 
one  but  to  a  number  of  animals.  In  a  physiological  experiment  of 
this  nature  it  must  not  be  expected  to  obtain  an  accuracy  compara- 
ble to  that  of  a  quantitative  chemical  analysis.  Sometimes  a  guinea- 
pig  will  not  die  when  inoculated  with  what  is  presumed  to  be  several 
times  the  minimum  fatal  dose.  Hence,  it  may  happen  that  out  of  a 
dozen  animals,  inoculated  with  the  minimum  fatal  dose,  perhaps  one 
or  two -may  eventually  recover.  The  resistance  of  the  animal  body 
is  not  a  constant  factor  and  for  that  reason  the  minimum  fatal  dose 
cannot  be  considered  as  an  absolute  result.  Moreover,  since  the 
strength  of  an  antitoxin  is  determined  by  testing  against  a  known 
amount  of  the  toxin  it  follows  that  the  former  result,  expressed  in 
immunity  units,  is  approximate  and  not  exact. 

Recognizing  the  difficulties  mentioned  above,  it  is  cus- 
tomary to  define  a  '  *  minimal  fatal  dose  "  as  that  amount  of 
toxin  which  will  usually  kill  guinea-pigs,  weighing  250  g., 
on  the  4th  day,  or  at  most  on  the  5th.  This  amount  may 
kill  a  very  susceptible  animal  in  1^-2  days.  If,  however, 
a  number  of  animals  die  in  less  than  two  days  it  is  evident 
that  the  quantity  employed  contains  more  than  one  mini- 
mum fatal  dose.  The  animal  should  be  weighed  and  the 
fatal  dose  per  250  g.  of  body  weight  calculated.  Thus,  if 


478  BACTERIOLOGY. 

it  is  desired  to  ascertain  the  dose  which  is  to  be  taken  for 
an  animal  weighing  300  g.  on  the  basis  of  0.05  c.c.  per  250 

:g.,  then: 

300  :  x  ::  250  :  0.05        x  =  0.06. 

Testing  of  Antitoxin. 

The  strength  of  an  antitoxic  serum  is  expressed  in  immu- 
nity units.  An  immunity  unit  may  be  denned  as  the  amount 
of  antitoxin  which  is  present  in  10  times  the  amount  of 
serum  that  is  just  sufficient  to  protect  a  250  g.  guinea-pig 
against  10  times  the  minimal  fatal  dose  of  toxin.  In  other 
words,  an  immunity  unit  will  theoretically  protect  against 
100  times  the  minimum  fatal  dose.  The  protection  extends 
not  merely  to  saving  the  life  of  the  animal  but  must  also 
prevent  local  swelling,  as  well  as  variation  in  temperature 
and  in  body- weight.  If,  for  example,  0.1  c.c.  of  serum 
protects  a  guinea  pig,  then  1  c.c.  of  that  serum  is  said  to 
contain  1  immunity  unit.  Again,  if  0.001  c.c.  protects  then 
1  c.c.  of  such  serum  contains  100  immunity  units.  Serum 
•can  be  prepared  of  such  strength  that  the  astonishingly 
small  amount  of  0.000,05  c.c.  suffices  to  protect  a  guinea- 
pig.  This  serum,  therefore,  contains  2000  immunity  units 
in  1  c.c. 

The  usual  method  of  testing  antitoxin  is  to  inject 
subcutaneously  into  several  guinea-pigs,  each  weighing 
about  250  g. ;  mixtures  of  10  times  the  minimum  fatal  dose 
of  toxin  and  variable  amounts  of  antitoxin.  The  several 
mixtures  are  made  up  to  the  same  volume  by  the  addition 
of  sterile  physiological  salt  solution  (0.75  per  cent.  NaCl). 
The  amount  of  antitoxin  (a)  which  is  just  sufficient  to  pre- 
vent any  ill  effects,  even  local  edema,  represents  iV  of  an 
immunity  unit.  Hence,  ^  represents  the  number  of  im- 
munity units  present  in  1  c.c.  of  the  serum. 


TESTING  OF   ANTITOXIN.  479 

The  method  of  determining  the  strength  of  an  antitoxic  serum 
can  be  shown  by  the  following"  example: 

The  minimum  fatal  dose  of  the  particular  toxin  employed  was 
found  to  be  0.02  c.c.  Hence,  0.2  c.c.  of  this  toxin,  when  injected  into 
each  guinea-pig,  per  250  g.  body-weight,  represents  10  times  the 
minimum  fatal  dose.  In  order  to  measure  out  this  amount  it  is  advis- 
able to  add  1  c.c.  of  the  toxin  to  4  c.c.  of  the  NaCl  solution.  1  c.c.  of 
this  dilute  toxin  (A)  represents  0.2  c.c.  of  the  original  poison. 

1  c.c.  of  toxin  A  corresponds  to  10  times  the  minimum  fatal  dose 
for  a  guinea-pig  weighing  250  g.  It  is  not  always  possible  to  obtain 
animals  that  will  have  exactly  the  same  weight.  They  should,  how- 
ever, weigh  as  close  to  this  amount  as  is  possible;  and,  a  correspond- 
ing correction  for  such  variation  should  be  made  when  measuring  out 
the  toxin  and  antitoxin.  Thus,  for  an  animal  weighing  300  g.  the  dose 
of  toxin  A  is  ascertained  from  the  proportion: 

300  :  x  :  :  250  :  1  x  =  1.2  c.c.  toxin  A. 

The  calculated  amounts  of  toxin  A  should  then  be  measured  out 
into  small,  sterile  Esmarch  dishes.  Eventually,  the  calculated  quan- 
tities of  serum  are  added  and  each  mixture  is  then  injected  into  the 
proper  guinea-pig. 

If  it  is  desired  to  ascertain  whether  the  antitoxin  possesses  the 
strength  claimed,  the  contents  of  the  bottle  should  be  drained  into  a 
sterile  Esmarch  dish.  By  means  of  an  accurate  pipette  the  volume 
of  the  serum  can  be  readily  determined.  If  the  bottle  is  said  to  con- 
tain 2000  I.  U.  and  7  c.c.  are  present  it  is  evident  that  each  c.c.  of  the 
serum  should  contain  at  least  286  I.  U. 

Suitable  dilutions  of  the  serum  are  now  made  in  order  to  facili- 
tate the  measuring  out  of  small  quantities.  Thus,  0.1  c.c.  of  the 
serum  is  added  to  9.9  c.c.  of  the  NaCl  solution  — -  dilution  A.  0.1  c.c. 
of  this  dilution  represents  0.001  c.c.  of  the  original  serum. 

A  second  dilution  may  be  prepared  by  adding  0.5  c.c.  of  dilution 
A  to  4.5  c.c.  of  the  NaCl  solution  —  dilution  B.  0.1  c.c.  of  this  dilu- 
tion represents  0.0001  c.c.  of  the  original  serum.  This  amount, 
according  to  the  definition  of  an  immunity  unit,  corresponds  to  1000 
I.  U.  Hence, 

0.25  c.c.  of  dilution  B  (0.00025  c.c.  serujn)  represents  400  I.  U. 
0.29  c.c.  of  dilution  B  (0.00029  c.c.  serum)  represents  345  I.  U. 
0.33  c.c.  of  dilution  B  (0.00033  c.c.  serum)  represents  303  I.  U. 
0.35  c.c.  of  dilution  B  (0.00035  c.c.  serum)  represents  286  I.  U. 

The  above  amounts,  it  will  be  understood,  refer  to  a  guinea-pig  weigh- 
ing 250  g.      Corrections  for  variations   from  this  weight  should  be 


480 


BACTERIOLOGY. 


made  as  in  the  case  of  the  toxin.     Thus,  if   the  250  g.  animal  is  to 
receive  0.25  c.c.  of  serum  B,  but  the  one  taken  weighs  300  g.,  then, 


300 


250  :  0.25 


x  =  0.30  c.c.  serum  B. 


The  corrected  amount  of  serum  B  is  measured  out  for  each 
guinea-pig"  and  added  to  the  similarly  corrected  amount  of  toxin  A. 
The  mixture  is  then  injected  subcutaneously .  The  temperature  and 
weight  should  be  taken,  and  the  local  effects  should  be  observed.  If  a 
given  injection  produces  no  ill  effect  it  is  evident  that  the  amount  of 
serum  injected  contains  at  least  the  corresponding  number  of  immuni- 
ty units.  It  may,  of  course,  contain  a  larger  number.  On  the  other 
hand,  if  the  animal  dies,  it  is  evident  that  the  serum  does  not  contain 
as  many  I.  U.  as  are  represented  by  the  dose  taken.  If  only  a  local 
swelling,  or  a  slight  scar  results  it  indicates  that  the  serum  is  nearly 
strong  enough  to  neutralize  the  toxin  injected. 

The  testing  of  an  antitoxin  will  be  rendered  more  clear  by  means 
of  the  following  table: 


CORRECTED   DOSE  FOR 

DOSE    FOR   250   G.            ||    £ 

H 

ACTUAL  WEIGHT. 

BODY-WEIGHT. 

S2 

NO 

X 

i 

RESULT 

0 

w 

TOXIN  A. 

SERUM  B. 

TOXIN  A. 

SERUM  B.     1 

*g 

is 

C.C. 

C.C. 

C.C. 

c.c.        1 

X 

1 

300 

1.2 

0.30 

1.0 

0.25 

400 

Died. 

2  • 

240 

0.96 

0.278 

1.0 

0.29 

345 

Slight  scar. 

3 

220 

0.88 

0.281 

1.0 

0.33 

303 

No  scar:  slight 
gain  in  weight. 

4 

260 

1.04 

0.364 

1.0 

0.35 

286 

Marked  gain 

It  is  evident  from  the  above  that  the  serum  contains  more  than 
286  I.  U.,  the  strength  claimed.  The  death  of  No.  1  indicates  that 
the  serum  has  less  than  400  I.  U.  per  c.c.  The  actual  amount  lies 
between  303  and  345. 

It  should  be  borne  in  mind  that  toxins  of  different 
origin  and  of  different  age  have  unequal  neutralizing 
power.  That  is  to  say,  the  amount  of  serum,  containing 
one  immunity  unit,  which  has  been  found  sufficient  to 
neutralize  100  times  the  minimum  fatal  dose  of  a  given  toxin, 
will  not  necessarily  neutralize  100  times  the  minimum  fatal 
dose  of  another  toxin.  This  difference  is  more  pronounced 
the  older  the  culture.  Thus,  a  culture  several  weeks  old 


TESTING  OF    ANTITOXIN.  481 

may  yield  a  toxin  of  which  an  amount  representing-  20  or  30 
times  the  minimum  fatal  dose  is  sufficient  to  neutralize  one 
immunity  unit.  Hence  in  testing-  antitoxins,  the  toxin7 
obtained  from  cultures  which  are  but  about  a  week  old 
should  be  employed. 

The  method  described  recently  by  Ehrlich  is  to  be  considered  as 
a  distinct  improvement  upon  the  ordinary  method  of  determining-  the 
strength  of  an  antitoxin.  It  is  based  upon  the  fact,  mentioned  above, 
that  the  neutralizing-  power  of  a  toxin  with  reference  to  a  serum  does 
not  depend  upon  the  number  of  minimum  fatal  doses  present.  Al- 
thoug-h,  theoretically,  1  I.  U.  should  neutralize  100  minimum  fatal 
doses  yet  it  has  actually  been  found  to  neutralize  anywhere  from 
16  to  108  doses.  This  great  variation  is  due  to  differences  in  the  com- 
position of  various  toxins.  According  to  Ehrlich  the  pure  toxin  be- 
comes converted,  to  a  greater  or  less  extent;  into  "  toxoids "  which, 
while  they  are  no  long-er  poisonous,  yet  possess  the  same  neutralizing 
power  as  the  original  toxin. 

Hence,  the  amount  of  toxin  employed,  when  testing  an  antitoxin, 
should  depend  not  upon  the  minimum  fatal  dose,  but  upon  its  neutral- 
izing power  with  reference  to  1 1.  U.  of  a  standard  test-serum.  Such 
a  standard  serum,  prepared  according  to  Ehrlich's  directions,  if 
available,  would  be  invaluable  for  comparisons  and  for  the  determin- 
ation of  the  actual  strength  of  an  antitoxin.  This  test-serum  is 
dried  in  vacuo  in  the  presence  of  phosphoric  anhydride,  and  is  then 
preserved  in  vacuum  tubes.  The  strength  of  the  serum  is  said  to 
remain  unaltered,  owing  to  the  absence  of  air  and  of  moisture. 

The  contents  (2  g.)  of  one  of  these  tubes  of  test-serum  is  dis- 
solved in  200  c.c.  of  a  mixture  of  equal  parts  of  glycerin  and  10  per 
cent.  NaCl  solution.  Inasmuch  as  the  original  serum  had  a  strength 
of  1700  I.  U.  it  follows  that  1  c.c.  of  this  solution  represents  17  I.  U. 

The  test-dose  of  toxin  is  to  be  ascertained  by  means  of  1  I.  U.  of 
this  standard  serum.  By  the  test-dose  of  toxin  is  understood  the 
amount  which,  mixed  with  1  I.  U.  and  injected  subcutaneously  into  a 
250  g.  guinea-pig,  will  cause  death  on  about  the  4th  day.  The  test- 
dose  can  be  readily  and  accurately  ascertained  by  adding  to  portions 
of  the  dilute  serum,  each  containing  II.  U.,  variable  amounts  of  the 
toxin  (30,  50,  70,  100  times  the  minimum  fatal  dose)  and  then  injecting 
these  mixtures  into  guinea-pigs. 

To  determine  whether  a  given  serum  has  an  advertised  strength 
a  portion  of  it  should  be  diluted  so  that  presumably  1  I.  U.  is  contained 

31 


482  BACTERIOLOGY. 

in  4  c.c.  The  test-dose  of  toxin  is  added  to  this  amount  of  serum  and 
the  mixture  is  then  injected  subcutaneously  into  a  250  g.  guinea-pig. 
If  the  trial  serum  actually  does  contain  1  I.  U.  death  will  not  result 
till,  as  in  the  above  determination  of  the  test-dose,  on  about  the  4th 
day.  If  the  animal  dies  on  the  2nd  or  3rd  day  it  is  evident  that  the 
amount  of  serum  taken  contains  less  than  1 1.  U.  and  that  the  original 
serum  is  of  less  strength  than  advertised.  If,  on  the  other  hand,  the 
animal  does  not  die  till  about  the  6th  day,  or  even  recovers,  it  follows 
that  the  amount  of-  serum  taken  contains  more  than  1  I.  U. 

In  the  latter  case  a  series  of  guinea-pigs  can  be  injected  with 
mixtures  of  the  test-dose  of  toxin  and  variable  amounts  of  the  dilute 
serum.  Eventually  a  dilution  of  the  serum  will  be  reached  which  will 
correspond,  as  indicated  above,  to  1  I.  U.  The  number  of  I.  U.  in  1  c.c. 
of  the  undiluted  serum  can  then  be  readily  calculated. 


Immunization    against  Diphtheria. 

The  diphtheria  toxin  prepared  according  to  the  direc- 
tions given  can  be  employed  for  immunizing  several  rabbits. 
The  animals  should  first  be  weighed  and  their  temperature 
taken.  For  the  first  injection  they  should  not  receive  more 
than  what  corresponds  to  the  minimum  fatal  dose  for  guinea- 
pigs.  Indeed,  this  amount  of  toxin  may  be  injurious  and  it 
is  advisable  to  add  to  the  toxin  a  drop  of  diluted  iodine  solu- 
tion (p.  288).  The  animals  can  tolerate  the  toxin,  thus 
modified,  better  than  if  it  were  used  direct.  The  tempera- 
ture and  weight  of  the  animals  are  taken  on  each  following 
day.  When  the  condition  of  the  animals  has  become  nor- 
mal, a  second  injection  is  made  and  this  is  repeated  until 
the  animals  cease  to  appreciably  react.  The  dose  of  toxin 
is  then  doubled  and  this  amount  with  a  little  iodine  is 
repeatedly  injected.  A  new  injection  should  not  be  given 
until  the  animals  have  fully  recovered  from  the  preceding  one. 
When  the  animals  shows  no  marked  results,  variation  in 
temperature  or  loss  in  weight,  after  the  injection  of  this 
amount  of  toxin  then  4  times  the  minimum  fatal  dose  may 
be  used,  mixed  as  before  with  a  little  iodine.  Eventually 


IMMUNIZATION   AGAINST  DIPHTHERIA.  483 

about  10  times  the  minimal  dose  mixed  with  iodine  should 
be  administered. 

The  animals  have  now  acquired  a  certain  resistance  to  the 
toxin  and  the  addition  of  iodine  should  be  omitted.  The 
dose  of  pure  toxin  should  be  small  for  the  first  injection, 
preferably  the  minimum  fatal  dose.  Several  injections  of 
this  amount  should  be  given  and  later  the  dose  may  be 
doubled  or  quadrupled.  The  injections  are  repeated  with 
the  same  or  gradually  increasing  doses  as  the  health  of  the 
animals  will  permit.  The  best  results  in  immunization  are 
always  obtained  by  proceeding  slowly.  The  animals  should 
be  given  plenty  of  time  to  recover  from  the  ill  effects  of  the 
last  injection. 

Eventually,  large  doses  of  toxin  can  be  tolerated  by  the 
experimental  animals.  The  fact  that  a  condition  of  immun- 
ity exists  can  be  easily  demonstrated  by  injecting  a  new 
untreated  rabbit  with  the  same  amount  of  toxin  as  is  given 
to  the  treated  animals. 

When  the  animals  have  been  immunized  to  such  an  ex- 
tent that  they  can  bear  a  large  dose  of  toxin,  say  50  or  100 
times  the  minimum  fatal  dose,  a  small  amount  of  blood  may 
be  drawn  and  its  antitoxic  properties  tested.  The  best 
procedure,  in  this  case,  will  be  to  draw  about  3  c.c.  of  blood 
from  the  jugular  vein  by  means  of  a  syringe  (p.  461),  and  to 
inject  this  at  once,  subcutaneously,  into  a  guinea-pig.  The 
latter,  as  well  as  a  control  animal  should  then  be  given  an 
injection  of  5  or  10  times  the  minimum  fatal  dose.  The  ani- 
mal should  survive,  and,  if  the  amount  of  antitoxin  is  suffi- 
cient, it  will  show  no  induration,  swelling  or  scar  at  the 
point  of  inoculation. 

The  injections  should  always  be  made  subcutaneously. 
The  toxin  should  be  made  up  to  a  convenient  volume  (1  or 
2  c.c.)  by  the  addition  of  physiological  salt  solution.  It  is 
hardly  necessary  to  add  that  all  the  instruments  and  glass- 
ware employed  must  be  sterile. 


484  BACTERIOLOGY. 

t 

Anti-Infectious  Serum. 

The  serum  of  an  animal  which  has  been  immunized  by 
repeated  injections  of  a  soluble  toxin,  as  in  the  case  of  diph- 
theria, possesses  antitoxic  properties.  That  is  to  say,  it 
contains  a  substance,  antitoxin,  which  in  some  way  neutral- 
izes or  renders  inert  the  soluble  poison  when  introduced 
into  the  body.  This  antitoxic  serum  should  be  sharply  dis- 
tinguished from  the  anti-infectious  serum  which  results  when 
an  animal  is  immunized  by  repeated  injections  of  certain 
living"  or  dead  bacteria.  The  defense  of  the  body  is  carried 
out  either  by  destroying  the  soluble  poison  that  is  being 
made  in  the  body,  or  by  destroying  the  bacteria  themselves. 
There  are,  therefore,  two  distinct  agencies  at  work. 

By  repeated  injections  of  dead  or  living  bacteria  the 
cells  are  taught  to  take  up,  or  otherwise  destroy,  the  solid 
invading  organisms.  The  serum  of  an  animal  thus  immun- 
ized possesses  the  property  of  stimulating  or  causing  the 
destruction  of  bacteria.  It  may  incidentally  possess,  more 
or  less,  antitoxic  action.  If,  for  instance,  the  pest  bacillus 
is  injected  subcutaneously  into  the  horse  the  organism  is 
localized  at  the  point  of  injection  and  may  grow  at  that 
point  for  some  time.  In  so  doing,  it  elaborates  poisonous 
products  which  now  induce  the  same  reaction  in  the  body 
as  if  they  were  injected  separately.  For  the  same  reason, 
the  blood  of  a  horse  immunized  with  living  diphtheria 
bacilli  is  antitoxic  rather  than  anti-infectious. 

The  anti-infectious  serum  has  been  especially  studied 
in  connection  with  cholera,  typhoid  fever,  pest  and  rouget. 
It  is  evident  from  what  has  been  said  that  a  given  organ- 
ism may  give  rise  to  serums  entirely  distinct  in  action  ac- 
cording as  the  soluble  poison  or  the  solid  cell  is  injected. 
Thus,  the  serum  of  an  animal  vaccinated  with  the  living  or 
dead  cholera  vibrio  will  protect,  in  even  extremely  minute 
amount,  against  inoculation  with  the  living  germ,  but 


ANTI-INFECTIOUS    SERUM.  485 

it  will   not   protect   against   the   injection  of   the    soluble 
toxin. 

The  antitoxic  serum  can  be  used  as  a  preventive,  or  as~ 
a  curative  agent  whereas  the  anti-infectious  serum  has  a 
preventive  value  only.     Even  this  action  is  manifest  only 
under  certain  experimental  conditions.    Thus,  the  anti-infec- 
tious cholera  serum,  which  protects  perfectly  against  sub 
cutaneous   or    intraperitoneal    injections    of    the    cholera 
vibrio,  is  of  no  value  if  the  organism  is  introduced  into  the 
intestinal  canal.      The   soluble  poison   elaborated   by  the 
vibrio   in  the  intestines  is    absorbed  and  can  be  counter- 
acted or  rendered  inert  only  by  an  antitoxic  serum. 

An  animal  that  has  recovered  from  an  attack  of  a  dis- 
ease or  has  been  rendered  immune  by  treatment  with  a 
germ  or  a  toxin  is  said  to  have  acquired  active  immunity. 
The  blood  of  an  actively  immunized  animal  is  capable,  even 
in  a  small  dose,  of  conferring  a  temporary  exemption  from 
that  disease.  This  is  designated  as  passive  immunity. 
Thus,  the .  blood  of  an  animal  actively  immunized  against 
the  soluble  diphtheria  toxin  will,  if  injected,  confer  passive 
immunity  to  man  or  animals.  In  other  words,  the  soluble 
toxin  or  the  virulent  germ  induces  active  immunity,  where- 
as the  antitoxic,  or  anti-infectious  serum  obtained  from 
such  actively  immunized  animal  induces  a  condition  of 
passive  immunity. 

The  organism  employed  for  immunizing-  purposes  should  possess 
a  maximum  degree  of  virulence.  This  can  be  obtained  by  repeated 
passage  from  animal  to  animal  (p.  278),  or  by  cultivation  in  collodium 
sacs  (p.  496),  alternating  with  passage  through  animals.  The  cultures 
should  be  grown  on  agar  and  should  not  be  more  than  20  or  24  hours 
old. 

When  it  is  desired  to  obtain  a  large  surface  growth  of  bacteria 
the  Roux  flask  shown  in  Fig.  69  will  be  found  extremely  useful.  100 
c.c.  of  agar  or  more  are  placed  in  the  flask  and  sterilized.  On  placing 
the  flask  in  a  horizontal  position  the  agar  solidifies  and  a  large  sur- 
face is  thus  obtained.  It  can  be  inoculated  by  introducing  a  few 
drops  of  the  culture  suspended  in  bouillon,  or  better  by  swabbing  the 


486  BACTERIOLOGY. 

culture  over  the  entire  surface.  The  growth,  as  a  rule,  can  easily  be 
removed  by  adding-  sterile  water,  and  then  gently  agitating  the 
liquid.  The  thick  suspension  can  then  be  poured  out  into  sterile 
flasks  or  drawn  up  into  sterile  bulb  pipettes  (p.  457). 

To  measure  out  a  dose  of  the  growth  Pfeiffer  employs  a  small 
loop  (Oese)  that  will  hold  about  2  mg.  of  the  material.     This  would 
correspond  to   a  loop  about  1  mm.    in  diameter.     A  loopful  of   the 
growth  is  stirred  up  into  1  c.c.  of  bouillon.    A  fraction  of  a  loop,  as  X> 
is  obtained  by  transferring-  1   loopful  to  4  c-c.  of 
bouillon  and  then  taking-  1  c.c.  of  the  suspension. 
For  the  preliminary  injections,  it  is  advis- 
able to  employ  dead  cultures.      The    suspension 
can  be  heated  for  1  hour  at  58-65°,  or  it  may  be 
shaken  up  with  a  few  drops  of  chloroform.     If 
living  cultures  are  employed,  the  dose  for  the 
first  injection  must  be  a  very  small  fraction  of 
the  minimum  fatal  dose.     A  record  must  be  kept 
of  the  temperature  and  weight  of  the  animal, 
and,  at  no  time,  must  an  injection  be  repeated 
suffaGc'e6?ult?r°eUsX  flask  for  before  the  temperature  and  weight  have  returned 
to  the  normal.     The  first  effect  of  an  injection  is 

to  -cause  a  slight  elevation  of  temperature  which  is  followed  by  a 
depression.  The  temperature  may  fall  to  30°  or  lower,  before  death 
occurs.  The  animal  likewise  loses  considerably  in  weight. 

Cholera. — Pfeiffer  immunized  guinea -pigs  by  injecting 
into  the  peritoneal  cavity  one-third  of  an  agar  culture, 
sterilized  by  chloroform  or  by  heating  at  65°.  Seven  to  ten 
days  later,  about  half  a  loopful  of  the  living  culture  is  in- 
jected in  the  same  manner.  After  a  like  interval,  one  loop- 
ful of  the  living  culture  is  injected;  then,  when  the  animal 
recovers  two  loopsful  can  be  given.  In  this  way  the  dose 
can  be  progressively  increased  till  lasting  immunity  is  ob- 
tained. Guinea-pigs  can  thus  be  immunized  so  as  to  with- 
stand 50  or  75  times  the  fatal  dose. 

When  the  guinea-pig  has  acquired  considerable  resist- 
ance a  large  dose  of  the  cholera  vibrios  should  be  injected 
into  the  peritoneal  cavity.  A  few  minutes  later,  a  drop  of 
liquid  should  be  removed  from  the  cavity  by  means  of  a 
drawn-out  capillary  tube  (Fig.  62  c),  and  examined  at  once 


ANTI-INFECTIOUS    SERUM,  487 

in  the  hanging-drop.  Many  of  the  cells  will  be  seen  to  be 
perfectly  motionless,  while  others  are  breaking  up  into 
granules.  A  drop  of  liquid  should  be  drawn  from  the  peri- 
toneal cavity  at  the  end  of  10,  20  and  30  minutes  after  the 
injection.  The  cells  are  first  immobilized;  they  then  break 
up  into  fragments,  become  granular  and  finally,  in  about  30 
minutes,  they  disappear  completely  "like  a  piece  of  sugar 
in  water."  This  is  "known  as  Pfeiffer's  reaction.  Exactly 
the  same  condition  will  be  observed  if  a  mixture  of  the 
serum  of  this  animal  and  of  the  virulent  cholera  vibrios  is 
injected  into  a  normal  guinea-pig.  If  the  vibrios  injected  do 
not  dissolve  and  disappear,  it  is  conclusive  evidence  that  they 
belong  to  a  different  species.  Furthermore,  it  may  be  stated 
that  the  cholera  vibrio  will  not  disappear  if  injected  by  itself 
into  a  guinea-pig,  whereas  harmless  vibrios  will  dissolve. 
The  serum  of  the  animal  thus  vaccinated,  as  stated 
above,  is  anti-infectious.  It  will  protect  against  the  living 
germ  but  not  against  the  soluble  poison.  If  the  cholera 
vibrio  is  planted  in  such  serum  it  is  not  destroyed;  it  may 
become  agglutinated,  but  Pfeiffer's  phenomenon  will  not  be 
seen.  In  other  words,  the  serum  is  neither  germicidal  nor 
antitoxic. 

The  anti-infectious  serum  as  indicated  is  not  germicidal 
in  vitro,  nor  is  it  antitoxic;  and  yet,  a  small  amount  is 
sufficient  to  save  an  animal  from  many  times  the  fatal 
dose  of  the  living  culture.  The  organisms  in  this  case  may 
be  totally  destroyed  in  less  than  30  minutes.  It  may  be, 
as ,  Pfeiffer  believes,  that  this  small  amount  of  serum  is 
converted  in  the  body  into  a  germicidal  substance  but  it  is 
more  probable  that  this  serum  acts  by  stimulating  the 
white  blood  cells  and  other  phagocytic  elements,  which 
then  give  off  the  peculiar  products  that  induce  the  Pfeiffer 
phenomenon. 

It  may  be  incidentally  stated  that  the  antitoxic  serum 
likewise  possesses  no  germicidal  properties.  It  is  supposed 


488  .         BACTERIOLOGY. 

to  neutralize  the  toxin  in  much  the  same  way  that  an  acid 
and  alkali  unite  and  neutralize  one  another.  While  this  is 
a  convenient  way  of  explaining-  the  action  of  antitoxin,  it  is 
probably  no  more  correct  than  it  would  be  to  assume  that 
the  anti-infectious  serum  is  germicidal.  Antitoxic  serum, 
like  anti-infectious  serum,  does  not  act  directly  but  it  stimu- 
lates the  cellular  defenses  of  the  body  to  activity.  The  cells 
of  the  body  by  preliminary  training-,  such  as  repeated 
poisoning-  with  a  toxin,  can  take  up  such  poison  or  give  off 
products  which  will  destroy  it.  If,  as  in  the  cholera  ex- 
periment above,  living-  cells  are  injected  then  the  phag-ocytes 
are  brought  into  action  either  directly,  or  by  their  germi- 
cidal  products.  In  either  case  immunity  always  depends, 
directly  or  indirectly,  upon  the  cellular  elements  of  the 
body. 

Pfeiffer's  phenomenon  is  not  limited  to  the  cholera 
vibrio  but  will  be  met  with,  under  like  conditions,  when  the 
Eberth  bacillus  or  other  org-anism  is  employed  for  immuniza- 
tion. It  is  of  great  value  as  a  means  of  differentiating-  the 
cholera  vibrio  from  other  similar  vibrios;  or  the  Eberth 
bacillus  from  the  colon  and  other  Eberth-like  bacilli.  The 
reaction  is  specific.  That  is  to  say,  the  anti-infectious  cholera 
serum  will  cause  the  destruction  of  cholera  g-erms  inside 
the  peritoneal  cavity  of  an  animal.  It  will  not  protect 
ag-ainst  other  vibrios  or  ag-ainst  the  Eberth  bacillus.  Simi- 
larly, the  anti-infectious  typhoid  serum  will  protect  ag-ainst 
the  Eberth  bacillus  but  not  ag-ainst  the  colon  or  pseudo- 
typhoid  bacteria. 

In  employing  this  test  it  should  be  remembered  that 
normal  serum  may  protect,  in  1  c.c.  dose,  against  the 
minimal  fatal  dose  of  the  cholera  or  typhoid  bacteria. 
Hence,  several  times  the  fatal  dose  should  be  employed  and 
a  control  test  should  be  made  with  a  like  amount  of  normal 
serum.  The  serum  of  convalescents  from  typhoid  fever  is 
not  antitoxic  but  is  anti-infectious  and  can,  therefore,  be 


ANTI-INFECTIOUS    SERUM.  489 

used  in  testing  the  genuineness  of  a  suspected  Eberth 
bacillus.  A  few  hundredths  of  a  c.c.  of  such  serum,  in- 
jected into  the  peritoneal  cavity,  will  protect  a  guinea-pig 
against  several  times  the  fatal  dose  of  Eberth  bacilli, 
whereas  0.5  c.c.  of  such  serum  will  not  protect  against  the 
colon  bacillus. 

Typhoid  fever. — The  Eberth  bacillus,  like  the  cholera 
vibrio,  colon  bacillus,  etc.,  does  not  give  rise  to  very 
poisonous  soluble  products  when  grown  on  ordinary  bouillon. 
A  soluble  toxin  has,  however,  been  recently  obtained  by 
cultivating  the  highly  virulent  germ  on  an  alkaline  medium 
prepared  by  digesting  the  spleen  with  pepsin.  With  such  a 
toxin  Chantemesse  has  been  able  to  obtain,  although  with 
difficulty,  an  antitoxic  serum. 

When,  however,  living  or  dead  Eberth  bacilli  are  in- 
jected subcutaneously  or  intraperitoneally,  a  condition  of 
immunity  is  established  and  the  serum  in  this  case  is  anti- 
infectious.  The  method  of  immunizing  the  guinea-pig  or 
rabbit  is  essentially  the  same  as  that  given  above  under 
cholera.  The  serum  of  a  guinea-pig  immunized  in  this  way 
has  about  the  same  preventive  action  as  that  of  a  con- 
valescent from  typhoid  fever.  The  rabbit  is  more  sensitive. 
The  virulence  of  the  Eberth  bacilli  will  vary  greatly,  thus, 
some  will  produce  death  in  a  guinea-pig  when  *V  of  a  loop- 
ful  is  injected  into  the  peritoneal  cavity.  Usually,  however, 
i  -  £  a  loopful  is  necessary.  2-3  loopsful  of  a  culture,  which 
has  been  killed  by  exposure  to  chloroform,  or  to  a  tempera- 
ture of  65°,  will  be  fatal  to  a  100  g.  guinea-pig  in  24  hours. 

The  defibrinated  blood  of  an  animal  that  has  died  of 
an  infectious  disease  may  be  employed  to  confer  immunity. 
Thus,  Toussaint  injected  into  sheep  3  c.c.  of  defibrinated 
anthrax  blood,  previously  heated  to  55°  for  10  minutes,  and 
obtained  immunity.  The  heart-blood  of  rabbits  dead  of 
swine-plague,  when  heated  to  54-58°,  will  protect  rabbits  in 


490  BACTERIOLOGY. 

a  dose  of  1.5  c.c.  introduced  subcutaneously  or  intraperi- 
toneally  (Selander).  In  the  latter  case,  Metchnikoff  has 
shown  that  the  serum  of  the  immunized  animal  has  neither 
antitoxic,  germicidal  nor  attenuating-  action.  It  will  not 
protect  against  the  soluble  toxin!  The  serum,  therefore, 
is  anti-infectious  and  stimulates  the  phagocytes  to  destroy 
the  bacterial  cells. 


Eisner's  Medium. 

Eisner's  medium ]  is  a  potato  gelatin  to  which  1  per 
cent,  of  KI  has  been  added.  It  is  prepared  according  to 
the  method  of  Holz.2  The  details  of  preparation  as  given 
by  these  authors  are1  very  meager  and  unsatisfactory.  The 
method  of  preparation  as  adhered  to  in  this  laboratory  fol- 
lows as  closely  as  possible  the  original  data. 

1,000  g.  of  potatoes  are  weighed  out.  The  potatoes  are 
brushed  clean  under  the  tap  and  cut  up  into  lumps  which 
are  then  placed  in  an  Enterprise  fruit-press  (No.  34).  The 
potato  comes  through  in  a  finely  mashed  condition.  A 
sausage  machine  is  not  as  useful  since  it  cuts  up  the  potato 
into  small  lumps,  and  when  in  this  condition  less  juice  can 
be  obtained  on  subsequent  squeezing.  The  finely  mashed 
potatoes  are  placed  in  muslin  and  squeezed  as  much  as  pos- 
sible. The  dark  colored  juice  is  saved.  The  potato  mass, 
wrapped  in  muslin,  is  then  placed  in  a  press  and  pressure 
applied.  The  liquid  thus  obtained  is  combined  with  the 
former.  About  400  c.c.  of  a  dark  liquid  is  thus  expressed 
out  of  1  kg.  of  potatoes. 

The  liquid  is  set  aside  in  the  ice-chest  for  24  hours, 
after  which  it  should  be  filtered.  Owing  to  the  presence  of 
extremely  small  granules,  the  liquid  can  not  be  readily 
filtered  through  paper.  It  is  advisable,  therefore,  to  filter 

1  Zeitschrift  fur  Hygiene  21,  p.  29,  1896. 

2  Ibid,  8,  p.  159,  1890. 


ELSNER'S  MEDIUM.  491 

through  absorbent  cotton  according  to  the  procedure  de- 
scribed on  p.  237.  When  this  filter  clogs  the  surface  layer 
of  muslin  should  be  carefully  removed  or  scraped  when  the 
filtration  will  recommence. 

10  per  cent,  of  gelatin  and  1  per  cent,  of  KI  are  added 
to  the  dark  liquid  which  is  gently  warmed  at  about  40° 
till  the  former  has  dissolved.  Portions  of  10  c.c.  of  the 
liquid  are  then  titrated  with  &  NaOH  (p.  155).  10  c.c.  of 
the  potato  extract  usually  requires  about  1.6  c.c.  of  •&  NaOH 
for  neutralization.  It  may,  however,  require  as  much  as 
3.2  c.c.  The  addition  of  gelatin  still  further  increases  the 
acidity.  The  gelatin  should  be  acid,  but  not  too  much  so.( 
The  acidity  of  10  c.c.  of  the  gelatin  should  not  require  more 
than  2.0  c.c.  of  TTJ  NaOH.  If  it  requires  more,  the  excess 
over  2  c.c.  (p.  354)  should  be  neutralized  by  adding  the  cor- 
responding amount  of  N  NaOH  to  the  remaining  gelatin. 
For  example,  if  10  c.c.  of  the  gelatin  requires  3.2  c.c.  T* 
NaOH,  then  the  excess  acidity,  above  2.0,  corresponds  to 
1.2  c.c  TNTT  NaOH.  Now,  if  the  volume  of  the  gelatin  meas- 
ures 350  c.c.  this  amount  will  require  42  c.c.  of  &  NaOH  = 
4.2  c.c.  of  N  NaOH,  in  order  to  reduce  the  acidity  to  the 
proper  point. 

10  :  1.2  ::  350  :  x         x  =  42  c.c.  of  &  NaOH. 

The  gelatin,  having  now  the  proper  degree  of  acidity; 
is  immersed  in  a  boiling  water-bath  for  about  ^  of  an  hour. 
The  soluble  proteins  coagulate  and  clarify  the  liquid.  This 
is  then  filtered,  filled  into  tubes,  and  sterilized  by  steaming 
for  15  minutes  on  each  of  three  successive  days.  As  a  rule, 
a  slight  precipitate  forms  in  the  tubes,  during  sterilization, 
but  this  does  not  interfere  with  the  usefulness  of  the 
medium. 

On  this  medium  the  Eberth  bacillus  yields  very  finely 
granular,  small,  bright  droplets  resembling  water.  On  the 
other  hand,  the  colon  bacillus  gives  rise  to  colonies  which 
are  large,  spreading,  more  strongly  granular  and  brown  in 
appearance.  A  constant  temperature  of  15-18°  should  be 


492  BACTERIOLOGY. 

maintained  (p.  179).     The  acidity  of  this  gelatin  is  such  as 
to  inhibit  the  development  of  the  B.  icteroides. 

It  has  been  proposed  to  utilize  the  fact  that  the  typhoid  colony 
tends  to  give  off  delicate  fibrils  from  its'periphery,  whereas  the  border 

•  of  the  colon  bacillus  is  sharply  defined.     To  normal  urine  which  has 
been  allowed  to  stand  for  a  couple  of  days  till  the  reaction  is  alka- 
line, 0.5  per  cent,  of  pepton  and  3-5  per  cent,  of  gelatin  are  added. 
The  mixture  is  heated  in  the  water-bath  for  an  hour,  filtered,  filled 
into  tubes  and  sterilized.     This  is  done  by  steaming-  for  15  and  10  min- 
utes on  the  first  and  second  days,  respectively. 

Within  24  hours  at  22°  the  colon  colonies  appear  as  round,  yellow-  - 
ish,  finely  granular  and  sharp  bordered  bodies,  whereas  the  typhoid 

•  colonies  are  surrounded  by  whorls  of  threads.     In  this  way,  it  is  said 
to  be  possible  to  readily  detect  the  typhoid  bacillus  in  the  urine  or 
f  eces  of  the  patient. 


Stoddart's  Medium. 

The  Eberth  bacillus,  as  a  rule,  is  more  motile  than  the 
colon  bacillus.  This  fact  may  be  used  to  good  advantage 
in  distinguishing1  between  these  organisms.  Direct  micro- 
scopical examination  or  the  staining  of  flagella  will  not  give 
a  satisfactory  indication  of  the  motility.  If,  however,  a 
soft  medium  is  used,  the  motile  organism  will  rapidly 
diffuse  throughout  the  medium  or  over  its  surface,  whereas 
the  non-motile  organism  forms  a  thick,  white,  non-spread- 
ing growth. 

A  liter  of  meat  extract  is  prepared  in  the  usual  way 
(p.  153);  10  g.  of  pepton  and  5  g.  of  NaCl  are  added,  and  the 
mixture  slightly  warmed  till  solution  results.  It  is  then 
divided  into  two  equal  portions. 

To  one  portion,  10  per  cent,  of  gelatin  is  added,  and, 
when  this  has  been  dissolved,  the  liquid  is  titrated  with 
&  NaOH.  An  excess  of  10  c.c.  of  N  NaOH  per  liter  is  ad- 
ded to  impart  the  desired  alkalinity.  This  and  the  subse- 
quent procedure  is  exactly  the  same  as  that  described  on 
p.  155. 


STOOD  ART'S    MEDIA.  493 

The  other  portion  of  500  c.c.  of  meat  extract  is  also 
titrated  and  an  excess  of  10  c.c.  of  N  NaOH  per  liter  is  ad- 
ded. The  liquid  is  weighed;  then  boiled  and  filtered.  To  the— 
clear  bouillon,  thus  obtained,  5  g.  of  finely  cut  agar  are 
added.  The  liquid  is  gently  boiled  till  the  agar  has  com- 
pletely dissolved,  when  it  is  weighed.  The  loss  in  weight 
is  compensated  by  the  addition  of  a  corresponding  amount 
of  distilled  water.  An  equal  volume  of  the  10  per  cent, 
gelatin,  prepared  as  above,  is  then  added  and  the  mixture 
is  sedimented,  as  in  the  case  of  agar  (p.  236),  at  50°  for  1-2 
hours.  It  is  then  filtered  through  the  absorbent  cotton 
filter  (p.  237),  and,  if  desired,  it  can  then  be  passed  through 
a  filter  paper. 

The  preparation  of  this  medium  can  be  simplified  by  heating-  the 
liter  of  meat  extract  till  the  soluble  proteins  coagulate.  To  the 
clear  filtrate  5  g.  of  agar  are  then  added  and  the  liquid  is  boiled  till  this 
has  dissolved.  50  g.  of  gelatin  are  then  dissolved  in  this  agar  solution- 
and  the  liquid  is  titrated.  To  the  amount  of  normal  alkali  necessary 
to  neutralize  the  medium  an  excess  of  10  c.c.  per  liter  is  added  to  im- 
part the  desired  alkalinity.  The  liquid  is  then  heated  in  a  water- 
bath  for  about  30  minutes,  allowed  to  sediment  at  50°,  and  finally 
is  filtered  as  above. 

It  is  evident  that  Stoddart's  medium  is  a  gelatin-agar 
which  contains: 

Gelatin,  5  per  cent.  Agar,  0.5  per  cent. 

Pepton,  1.0  per  cent.  Salt,  0.5  per  cent. 

The  medium  is  filled  into  large  test-tubes,  in  portions 
of  about  10  c.c.,  and  sterilized  by  exposure  to  steam  for  15 
minutes  on  each  of  three  successive  days. 

The  method  of  using  the  medium  is  as  follows:  It  is  poured  out 
into  wide  (7  cm.)  Esmarch  dishes,  steamed  and  allowed  to  solidify  in  a 
horizontal  position.  The  organism  to  be  tested  is  touched,  by  means 
of  a  platinum  wire,  to  the  center  of  the  surface  of  the  medium.  The 
dishes  thus  prepared  are  placed  over  some  water  in  a  moist  chamber. 
The  latter  is  placed  in  a  horizontal  position,  in  the  incubator  at  35°.  Ins, 
about  18  hours,  the  Eberth  (and  Sanarelli)  bacilli  will  spread  over  the-  • 


494  BACTERIOLOGY. 

entire  surface  forming  a  transparent  scarcely  visible  growth.  The 
non-motile  colon  bacilli  ( Emmerich,  Havelburg),  will  form  a  small  white 
•colony  on  the  surface  without  any  diffusion.  .The  motile  colon  bacilli 
will  diffuse,  sometimes  as  rapidly  as  the  Eberth  bacilli,  but  unlike  these 
the  growth  will  be  opaque  and  easily  visible- 


Hiss'    Tube    Medium. 

This  is  similar  in  composition  to  the  above  and  contains 
in  addition  1  per  cent  of  glucose.  5  g.  of  Liebig's  meat  ex- 
tract, 5  g.  of  NaCl  and  5  g.  of  agar  are  added  to  1000  c.c.  of 
water.  The  mixture  is  heated  till  the  agar  is  dissolved. 
The  water  lost  by  evaporation  is  then  replaced  and  8  per 
cent,  of  gelatin  is  added.  When  this  has  dissolved  the  solu- 
tion is  titrated.  It  is  left  acid  but  the  acidity  is  reduced  by 
addition  of  N  NaOH,  so  that  it  corresponds  to  15  c.c.  of  nor- 
.  mal  acid  per  liter.  The  liquid  is  then  cooled  to  60 D  and 
cleared  by  adding  the  white  of  an  egg,  previously  beaten  in 
about  25  c.c.  of  water.  The  liquid  is  finally  boiled  for  a 
few  minutes  and  iO  g.  of  glucose  are  added.  It  is  then  sedi- 
mented  at  50°,  filtered  through  absorbent  cotton,  and  then 
through  paper  if  desired.  The  clear  filtrate  is  finally  filled 
into  tubes  and  sterilized. 

Stab  cultures  of  the  organisms  to  be  tested,  are  made 
in  this  medium.  The  colon  bacilli  and  B.  icteroides  give 
rise  to  gas  bubbles,  whereas  the  Eberth  bacillus  does  not 
produce  gas.  Gas  formation  will  be  observed  most  satis- 
factorily in  about  8  or  10  hours  after  the  tubes  have  been 
placed  in  the  incubator.  The  Eberth  and  the  motile  colon 
bacilli  diffuse  through  the  medium,  whereas  the  growth  of 
the  non-motile  colon  and  Eberth  bacilli  will  be  confined  to 
the  line  of  inoculation. 

The  difference  in  the  transparency  of  the  diffused 
growths  of  the  Eberth  and  colon  bacilli  as  observed  on 
Stoddart's  medium  is  not  seen  in  this  tube  medium.  It  is  a 
convenient  method,  however,  of  demonstrating  whether  an 


USCHINSKY'S   MEDIUM.  495 

organism  can  produce  gas  and  a  diffused  growth.  The 
latter  is  an  excellent  indication  of  the  presence  of  motion. 
The  Hiss  medium  can  be  modified  by  substituting^— 3U 
per  cent,  of  lactose  for  the  glucose,  and  by  rendering  the 
medium  alkaline  and  then  adding  litmus.  The  medium  thus 
modified  will  indicate  acid  and  gas  production  as  well  as 
diffusion. 

Uschinsky's  Medium. 

As  seen  from  the  formula,  this  is  an  artificial  medium 
made  up  of  simple  chemical  compounds  and  wholly  free 
from  protein  matter.  The  organisms  planted  on  this  medium 
are  therefore  obliged  to  build  up  their  protoplasm  from 
simple  organic  and  inorganic  bodies.  The  medium  has  been 
of  great  value  in  the. study  of  the  products  produced  by  the 
bacterial  cell.  It  possesses  the  following  composition: 

Water,  1000  parts,  Magnesium  sulphate,  0.2 — 0.4  part, 

Glycerin,  30-40  parts,  Di-potassium  phosphate,  2-2.5  parts, 

Sodium  chloride,  5-7  parts,  Ammonium  lactate,  6-7  parts, 

Calcium  chloride,  0.1  part,  Sodium  asparaginate,  3-4  parts. 

On  the  medium  prepared  as  above,  contrary  to  Uschin- 
sky,  the  typhoid  (and  Sanarelli)  bacillus  will  not  grow, 
whereas  the  colon  bacillus  yields  an  excellent  growth:  The 
medium,  therefore,  can  be  used  to  good  advantage  in  differ- 
entiating between  these  organisms. 

Fraenkel  has  simplified  the  above  solution  by  elimina- 
ting those  constituents  which  are  not  necessary  for  the 
growth  of  bacteria.  This  modified  solution  contains  5  g.  of 
NaCl,  2  g.  of  potassium  di-phosphate,  6  g.  of  ammonium 
lactate  and  4  g.  of  sodium  asparaginate.  Asparagin  may 
be  substituted  for  the  latter  and  the  neutral  phosphate  of 
sodium,  for  the  phosphate  of  potassium,  whereas  the  sodium 
chloride  may  be  omitted.  Fraenkel  observed  that  in  this 
medium  the  Eberth  bacillus  scarcely  developed,  whereas 
the  colon  bacillus  gave  a  rapid,  strong  growth. 


496  BACTERIOLOGY. 

A  1  per  cent,  solution  of  ammonium  chloride  and  of  gly- 
cerin, according-  to  Fischer,  likewise  differentiates  the  colon 
and  typhoid  bacilli.  The  former  can  live  on  nitrogen  in  the 
form  of  ammonia,  whereas  the  latter  is  unable  to  do  so. 
The  typhoid  bacillus  may  give  a  slight  growth  in  solutions, 
which  contain  nitrogen  in  the  amido  combination,  but  it  is 
especially  dependent  upon  the  nitrogen  of  protein  matter 
(pepton). 

Collodium  Sacs. 


The  usual  method  of  increasing  the  virulence  of  an  organ- 
ism is  by  successive  passage  through  animals  as  indicated  on 
p.  278.  This,  indeed,  has  been  the  only  procedure  until  the 
recent  introduction  of  the  collodium  sac  method  by  Metch- 
nikoff,  Roux  and  Salimbeni.  In  the  hands  of  the  French 
investigators  this  method  has  yielded  remarkable  results, 
notably  in  the  study  of  cholera,  pest,  pleuro-pneumonia 
and  tuberculosis. 

The  principle  of  the  method  consists  in  planting  the 
organism  in  a  small  collodium  sac,  which  is  then  sealed 
hermetically  and  placed  within  the  peritoneal  cavity  of  an 
animal.  Under  these  conditions  diffusion  takes  place.  The 
bacterial  products  pass  out,  whereas  the  albuminous,  highly 
nutritive  fluids  of  the  living  body  pass  into  the  sac.  The 
organism  is  thus  enabled  to  grow  in  the  fluids  of  the  living 
body  unaffected,  however,  by  phagocytes.  As  a  result,  it 
grows  luxuriantly  and  is  markedly  increased  in  virulence. 
The  growth  is  milky  in  appearance  and  the  bacteria  are 
vastly  more  abundant  than  in  ordinary  liquid  media. 

The  method  involves  the  most  delicate  bacteriological 
technique,  and,  inasmuch  as  the  details  of  the  process  have 
not  been  heretofore  published,  the  several  steps  will  be 
described  as  minutely  as  possible. 

The  collodium  on  the  market  varies  in  composition  according-  to 
the  kind  of  nitro-celhilose  employed.  Hence  every  collodium  cannot 


COLLODIUM  SACS.  497 

be  used  in  making-  sacs.  This  is  notably  true  of  celloidin  solutions. 
The  film  of  collodium  must  possess  a  certain  degree  of  elasticity, 
otherwise  it  cannot  be  slipped  off  the  tube  on  which  it  is  deposited. 

The  tubes  should  be  preferably  made  of  yellow  glass  and 
should  be  about  320  mm.  long.  The  end  should  be  perfectly  rounded 
off,  like  that  of  a  test-tube.  These  tubes  can  be  obtained  having-  a 
diameter  of  14,  18,  25  or  30  mm.  The  18  mm.  tube  is  the  most  con- 
venient one  for  ordinary  purposes. 


FIG.  70.    The  rolling  of  collodium  sacs.    The  beaker  contains  three  sacs. 

The  collodium  is  placed  in  a  small  glass  cylinder,  about  4  cm.  in 
diameter  and  9  cm.  hig-h.  This  is  inclined  in  the  manner  indicated  in 
Fig-.  70.  The  clean,  dry  glass  tube  is  inserted  into  the  collodium  and 
and  slowly  rotated.  Care  must  be  taken  to  avoid  touching-  the  walls 
of  the  cylinder.  The  tube  may  be  rested,  at  the  desired  angle,  on  the 
lower  jaw  of  a  retort  clamp.  From  time  to  time,  the  tube  is  with- 
drawn almost  completely  out  of  the  collodium  and  rolled  in  the  air. 
When  the  tube  is  returned  to  the  liquid  another  coat  of  collodium  is 
deposited.  By  repeating-  this  several  times  a  g-ood  layer  of  collodium 
is  deposited  on  the  glass.  It  may  happen  that  while  the  collodium 
is  of  the  proper  thickness  above,  where  it  has  been  repeatedly  ex- 
posed to  the  air,  yet  only  a  very  thin  layer  will  cover  the  rounded  end 
of  the  tube  since  this  has  remained  continuously  in  the  liquid.  This 
difficulty  can  be  overcome  by  completely  withdrawing-  the  tube  out  of 
the  collodium,  rotating-  a  few  moments  in  the  air  and  then  returning 
it  slowly  to  the  liquid.  This  should  be  done  slowly  and  at  as  much  of 
an  angle  as  possible  to  prevent  the  formation  of  air-bubbles  in  the 
wall  of  the  sac.  Minute  air-bubbles  do  not  impair  the  efficiency  of 
the  sac  but  large  ones  are  liable  to  burst  during  the  subsequent  pro- 
cess of  sterilization.  After  the  tube  has  been  taken  out  and  returned 
to  the  collodium  three  times,  it  will  usually  have  formed  upon  its 
surface  a  sufficiently  thick  layer  of  collodium. 

The  tube  is  then  rotated  slowly  in  the  air  to  allow  the  collodium 
to  partially  set.  By  proper  rotation  the  liquid  can  be  prevented  from 


498  BACTERIOLOGY. 

gathering  in  droplets.  The  turning  in  the  air  is  continued  till  the 
collodium  layer  is  no  longer  sticky  and  is  not  too  soft.  It  must  not  be 
exposed  to  the  air  till  it  becomes  too  dry,  inasmuch  as  it  would  then 
be  impossible  to  remove  it  from  the  glass  tube.  A  little  practice  will 
enable  one  to  tell,  by  the  touch  of  the  finger,  when  the  exposure  to 
air  has  been  sufficient. 

"The  coated  end  of  the  tube  is  then  immersed  in  a  beaker  of  dis- 
tilled water  and  rotated  in  this  for  several  minutes.  Tne  touch  of  a 
finger  will  again  indicate  when  the  desired  hardness  has  been  obtained. 
The  tube  is  then  withdrawn  and  the  sac  is  ready  to  be  peeled.  The 
collodium  layer  is  cut  circularly,  near  the  upper  end,  and  the  irregu- 
lar border  is  removed.  With  a  pair  of  forceps  or  with  the  finger-nail 
the  edge  of  the  collodium  layer  is  bent  back  on  itself.  When  the 
upper  portion  of  the  collodium  tube  has  been  turned  "inside  out",  the 
sac  can  be  drawn  off  the  tube  like  a  glove  off  a  finger.  The  tube 
is  grasped  over  the  turned  portion  of  the  sac  between  the  thumb  and 
first  two  fingers.  By  gently  drawing  on  this  portion  the  sac  is  slowly 
everted 

The  sac  thus  prepared  should  be  sufficiently  firm  so  as  not  to  col- 
lapse. It  is  then  filled  with  distilled  water  and  placed  in  the  beaker 
of  water  for  15  minutes  or  longer. 

The  next  step  is  to  prepare  a  glass- tube  which  will  enable  one  to 
hermetically  seal  the  sac.  The  author  employs  for  this  purpose  test- 
tubes  which  are  2  or  3  mm.  less  in  diameter  than  the  sac  itself.  The 
horizontal  flame  of  the  blast-lamp  is  directed  against  the  test-tube  at 
a  point  about  4  cm.  from  the  bottom.  The  tube  is  slowly  rotated  in 
the  flame  and  as  the  glass  softens  a  constriction  results.  When  cool 
the  rounded  end  of  the  tube  must  be  cut  off.  A  scratch  is  made  with 
a  file  at  about  1.5  cm.  below  the  constriction.  A  piece  of  burning 
charcoal  is  then  applied  to  the  end  of  the  scratch.  By  gently  breath- 
ing on  the  charcoal  this  can  be  kept  aglow  till  the  glass  cracks.  Usu- 
ally the  crack  extends  completely  around  the  tube,  but  if  it  does  not 
the  crack  can  be  led  around  by  holding  the  glowing  coal  before  it.  A 
hot  glass  rod  can  be  used  instead  of  the  charcoal.  The  cut  end  of  the 
tube  is  then  heated  in  the  flame  till  the  border  is  rendered  perfectly 
smooth.  The  tube  thus  prepared  has  the  appearance  shown  in  Fig. 
70  a.  A  number  of  these  tubes  should  be  prepared  and  kept  on  hand. 

When  a  large  sac  is  to  be  used  it  is  liable  to  break  after  it  is 
placed  in  the  abdominal  cavity.  This  defect  is  overcome  by  placing 
the  sac  in  a  wide  tube  which  is  freely  perforated.  The  following 
•modification  made  by  the  author  can  be  employed  (to  advantage  in 
place  of  the  usual  method.  In  this  procedure  openings  are  blown  into 


COLLODIUM    SACS. 


499 


a  test-tube  and  this  is  slipped  inside  of  the  sac.  The  first  opening- 
should  be  made  in  the  bottom  of  the  test-tube.  A  small  narrow  flame 
is  directed  against  the  bottom,  and,  when  the  glass  has  softened,  iTls 
touched  with  a  piece  of  drawn-out  glass-tubing  which  is  .then  with- 
drawn. The  glass  adheres  and  is  drawn  out  into  a  thin  capillary.  This 
is  then  broken  off  at  about  0.5  cm.  from  the  test-tube  by  a  gentle  tap  with 
the  glass-tube.  The  flame  of  the  blast-lamp  is  then  directed  against 
the  opening  thus  made.  The  broken  edges  sink  to  the  level  of  the 
tube  and  a  round  opening  results.  In  this  way  a  large  number  of  holes 
can  be  blown  into  the  lower  end 
of  the  tube  which  is  then  con- 
stricted in  the  manner  already 
described.  The  finished  perfor- 
ated tube  has  the  appearance 
shown  in  Fig.  71  e. 

The  next  step  is  to  attach 
the  collodium  sac  to  either  of 
these  constricted  tubes.  The 
open  end  of  the  sac  is  trimmed 
square  with  scissors.  The  sac  is 
then  placed  between  filter-paper 
and  dried  ~by  the  application  of 
gentle  pressure.  It  is  especially 
desirable  to  have  the  inside  of 
the  neck  of  the  sac  perfectly  dry. 
The  constricted  tube  is  then  in- 
serted into  the  sac  up  to  where 
the  constriction  begins.  This  tube 
and  sac  are  now,  carefully  and 
cautiously,  rotated  in  a  horizontal 
position  over  a  very  small  narrow  flame.  The  modified  Bunsen  burner 
provided  with  a  pilot  light  is  especially  useful  for  this  purpose.  The 
tube  and  sac  are  "held  at  a  distance  of  3-5  cm.  above  the  narrow  flame. 
The  collodium  contracts  down  upon  the  glass  and  forms  a  perfect  con- 
nection. To  make  this  absolutely  tight  the  collodium,  over  the  glass, 
should  be  rubbed  with  a  heated  rod.  In  the  absence  of  the  burner 
mentioned,  a  hot  glass  rod  passed  repeatedly  over  the  end  of  the  sac 
will  cause  this  to  adhere  to  the  glass. 

A  silk  thread  is  then  wrapped  over  this  portion  of  the  sac;  and, 
finally  it  is  thoroughly  coated  with  collodium  and  allowed  to  dry. 
The  object  of  the  silk  thread  is  to  enable  one,  later  on,  to  firmly  hold 
the  sac  in  a  pair  of  forceps.  The  sac  is  now  filled  with  distilled  water 
and  the  upper  end  of  the  tube  is  closed  with  cotton.  The  tubes  thus 


FIG.  71.  The  preparation  of  collodium  sacs. 
a — Test-tube,  constricted  and  cut;  b — The  sac 
attached  to  preceding  and  filled  with  water; 
c — Same  in  test-tube  on  foot,  in  water,  and 
sterilized;  e — Test-tube,  constricted  and  per- 
forated; d—  The  same  covered  with  sac. 


500  BACTERIOLOGY. 

prepared  (6  and  d)  are  then  placed  in  distilled  water  in  a  wide  test-tube 
on  foot  (Fig".  71  c).  The  sacs  are  now  sterilized  by  exposure  to  steam 
for  15  minutes,  on  each  of  three  successive  days;  'Or,  they  may  be 
sterilized  in  an  autoclave  at  115°  for  15  minutes.  In  this  condition 
the  sacs  can  be  preserved  for  several  weeks. 

The  filling1  of  the  sac  with  water  or  bouillon,  and  the  withdrawal 
of  that  present,  is  done  by  means  of  a  bulb  pipette  (Fig-.  61  e).  This 
should  be  bent  at  right  angles,  just  below  the  bulb.  The  tip  of  the 
pipette  is  cut  off  and  the  end  is  flamed.  Special  care  must  be  taken 
to  heat  the  cut  end  of  the  pipette  till  the  sharp  edges  are  rounded 
off. 

The  sac  is  now  ready  for  the  final  steps — inoculation,  sealing  and 
placing  in  the  abdominal  cavity.  The  cotton  is  removed  and  the 
mouth  of  the  test-tube  is  then  flamed.  The  narrowed  end  of  the  ster- 
ile pipette,  prepared  as  just  mentioned,  is  inserted  through  the  con- 
stricted portion  of  the  test-tube  into  the  sac.  The  water  is  now  care- 
fully drawn  up  into  the  bulb.  The  pipette  is  then  withdrawn  and  the 
cotton  plug  replaced.  The  water  is  blown  out  of  the  pipette  which  is 
then  filled  with  the  bouillon  that  has  been  inoculated  with  the  organ- 
ism to  be  tested.  The  pipette  is  again  inserted  into  the  sac  and 
this  is  now  filled  up  to  the  threaded  portion,  with  the  inoculated 
bouillon. 

Nothing  now  remains  but  to  seal  the  sac.  For  this  purpose,  it  is 
taken  out  of  the  wide  test-tube  and  the  constricted  portion  is  wiped 
dry  with  sterile  filter-paper.  The  sac  is  then  wrapped  in  a  piece  of 
sterile  paper  which  should  not  project  above  the  silk  thread.  One 
can  thus  grasp  the  glass  end  of  the  sac  without  causing  contamina- 
tion. A  very  narrow  flame  is  then  directed  against  the  constricted 
tube  till  it  is  sealed.  The  sac  is  held  in  the  sterile  paper,  avoiding 
unnecessary  pressure,  until  the  glass  cools.  The  glass  end  is  then 
coated  several  times  with  collodium,  and,  when  this  has  dried,  the  sac 
is  picked  up  by  a  pair  of  sterile  forceps  and  placed  in  a  sterile  dish 
(Fig.  43,  p.  265).  It  is  now  ready  to  be  inserted  into  an  animal. 

The  guinea-pig  or  rabbit  is  usually  used  for  sac  experiments, 
although  other  animals  may  be  employed.  The  animal  is  placed  on  a 
holder  (Fig.  45,  p.  268),  and  the  hair  over  the  abdomen  is  removed 
with  a  pair  of  curved  scissors.  The  surface  is  then  soaped  and 
shaved,  after  which  it  is  washed  with  alcohol  and  mercuric  chloride. 
A  piece  of  filter-paper  soaked  in  mercuric  chloride  is  then  spread 
over  the  prepared  surface.  The  animal  is  now  anesthetized  with 
ether.  An  incision,  about  3-5  cm.  long,  is  made  through  the  skin  of 
the  upper  part  of  the  abdomen.  A  similar  incision  is  made  on  one 


COLLODIUM  SACS.  501 

side  of  the  linea  alba,  through  the  abdominal  wall  to  the  peritoneum. 
A  grooved  sound  is  then  introduced  into  the  cavity  and  the  periton- 
eum is  opened.  The  abdominal  wall  on  each  side  of  the  incision  is 
seized  with  a  pair  of  pressure  forceps,  so  that  by  crossing  the  forceps 
the  cavity  can  be  closed. 

By  means  of  a  pair  of  forceps,  the  sac  is  picked  up,  at  the 
threaded  portion,  and  inserted  into  the  abdominal  cavity.  The  ab- 
dominal wall  is  then  sewed  by  means  of  a  curved  needle.  An  assist- 
ant should  hold  the  forceps  so  as  to  bring  the  cut  edges  together  and 
have  the  walls  on  a  stretch.  When  the  abdominal  wall  has  been 
closed  the  ends  of  the  silk  thread  are  cut  off  and  the  wound  disin- 
fected with  a  piece  of  filter-paper  soaked  in  mercuric  chloride.  The 
skin  is  then  sewed  in  like  manner.  The  surface  about  the  wound  is 
disinfected,  then  washed  with  absolute  alcohol  and  dried  with  sterile 
filter-paper.  A  few  tufts  of  absorbent  cotton  are  spread  over  the 
incision,  after  which  collodium  is  applied  and  smoothed  down  with 
forceps  or  knife.  When  this  has  thoroughly  dried  the  animal  can  be 
returned  to  its  cage. 

The  sac  is  allowed  to  remain  in  the  animal  for  from  3  to  5  days 
although,  in  special  cases,  it  may  remain  for  several  months.  When 
it  is  desired  to  remove  the  sac  the  animal  is  placed  under  a  bell-jar 
and  ether  or  illuminating  gas  is  introduced.  When  the  animal  is  dead 
it  is  spread  out  on  a  board  (see  p.  276)  and  opened.  The  sac  will 
usually  be  found  buried  among  the  intestines  and  surrounded  by  ad- 
hesions. It  should  be  carefully  removed  and  the  fibrinous  covering 
stripped  off.  It  is  then  placed  in  the  sterile  test-glass  (Fig.  43),  with 
the  glass  end  down.  A  hot  glass  rod  or  searing  iron  (Pig.  48  a,  p.  275), 
is  then  applied  to  the  end  of  the  sac  and  an  opening  is  thus  burned 
through.  The  milky  contents  of  the  sac  are  then  drawn  up  into  a 
sterile  bulb  pipette.  Transplantations  can  be  made  to  the  various 
nutrient  media  or  another  sac  may  be  inoculated.  The  end  of  the 
pipette  can  then  be  sealed  and  the  liquid  preserved  for  subsequent 
examinations. 

It  is  understood,  of  course,  that  all  the  instruments,  etc.,  used  in 
the  operation  must  be  sterile.  They  are  sterilized  by  boiling  in  a 
saturated  solution  of  borax  (Fig.  48  6,  p.  275).  The  hands  of  the  oper- 
ator should  be  thoroughly  scrubbed  with  soap  and  water  and  then  im- 
mersed for  some  minutes  in  mercuric  chloride  solution. 


502  BACTERIOLOGY. 


Inoculation  for  Rabies. 

The  sac  method  of  culture,  as  described,  is  not  only  use- 
ful in  increasing  the  virulence  of  an  organism,  but  it  possibly 
affords  a  means  of  growing  organisms  that  have  hitherto 
resisted  all  known  methods  of  cultivation.  It  has  already 
enabled  the  isolation  of  the  microbe  of  pleuro-pneumonia— 
an  organism  which  is  considerably  smaller  than  any  known 
representative  of ,  the  group  of  bacteria.  Undoubtedly,  the 
cause  of  other  diseases  such  as  small-pox,  scarlet  fever, 
rabies,  etc.,  will  be  found  to  belong  to  this  class  of  extremely 
minute  microbes. 

The  cause  of  rabies  has  not  as  yet  been  discovered,  but 
it  is  known  to  reside  especially  in  the  central  nervous  sys- 
tem. It  becomes  important,  at  times,  to  confirm  the  diag- 
nosis of  rabies  by  injecting  a  portion  of  the  spinal  cord  of 
the  suspected  animal  under  the  dura  mater  of  a  rabbit.  For 
this  reason,  the  following  method  of  inoculation,  as  prac- 
tised at  the  Pasteur  Institute  is  given: 

The  rabbit  is  fastened,  lying-  on  its  abdomen,  to  a  holder  or  to  a 
table  by  means  of  leather  thongs  or  stout  cords,  and  is  anesthetized 
with  ether.  The  head  of  the  animal  should  be  turned  toward  a  win- 
dow. The  excess  of  hair  is  removed  from  the  surface  of  the  skull 
which  is  then  moistened  with  carbolic  acid  or  with  lysol.  An  incision 
about  3  cm.  in  length  is  then  made.  One  flap  of  the  skin  is  raised  by 
a  pair  of  forceps  and  a  jaw  of  a  wire  speculum  is  inserted.  This  is 
done,  likewise,  to  the  other  side  and  the  speculum  is  then  distended. 
A  trephine  is  applied  to  the  exposed  skull  on  one  side  of  the  median 
line.  The  button,  about  6-8  mm.  in  diameter  is  picked  up  and  re- 
moved by  means  of  a  hooked  needle  or  tenaculum. 

A  portion  of  the  suspected  medulla  or  cord  has  previously  been 
rubbed  up  by  means  of  a  glass-rod  with  water  or  bouillon  in  a  test- 
glass  (Fig.  43,  p.  265).  The  suspension  is  drawn  up  into  a  syringe  and  1 
or  more  drops  are  injected  under  the  dura  mater.  A  few  drops  of  5 
per  cent,  carbolic  acid  solution  are  applied  to  the  surface  and  are  then 
carefully  removed  with  sterile  filter-paper.  This  is  repeated  several 
times,  and  finally,  after  drying  the  surface  thoroughly,  the  speculum 


INOCULATION  FOR  RABIES.  503 

is  removed.  The  wound  in  the  skin  is  then  closed  by  a  couple  of  silk 
sutures,  for  which  purpose  the  Reverdin  needle  is  usually  employed. 
The  incision  is  finally  covered  with  cotton  and  collodium  (p.  501). 

In  the  case  of  rabies,  the  rabbit  will  usually  die  in  about  14  to  20 
days.  By  repeated  successive  passage  the  virus  can  be  increased  in 
virulence  so  that  it  will  kill  invariably  in  10  days.  The  symptoms  of 
rabies  are  manifested  after  about  the  6th  day.  The  animal  becomes 
apathetic,  the  temperature  falls  considerably  below  the  normal,  and 
the  respiration  is  decreased.  The  loss  of  coordination  of  the  hind 
legs  is  characteristic  for  paralytic  rabies. 

The  cord  and  brain  of  the  dead  animal  should  be  exposed  and 
examined  by  staining,  and  by  the  culture  method  for  bacteria.  Similar 
examinations  should  be  made  of  the  heart-blood  and  internal  organs. 
The  absence  of  pathogenic  bacteria  must  be  established  in  order  to 
justify  the  conclusion  that  the  death  was  due  to  rabies. 

The  post-mortem  in  this  case  is  carried  on  as  follows:  The  ani- 
mal is  fastened  on  a  tray  or  board  and  an  incision  is  made  with  a  pair 
of  scissors  from  the  top  of  the  skull  to  the  root  of  the  tail.  The  lower 
end  of  the  incision  is  prolonged  towards  each  hind  extremity.  By 
means  of  a  knife  the  skin  is  then  wholly  removed  from  the  back  and 
side  of  the  animal,  and  also  from  over  the  skull,  the  ears  being  cut  off 
close  to  the  bone.  Beginning  at  the  base  of  the  skull  the  muscles  of 
the  neck  are  then  removed  by  a  pair  of  dull-pointed  scissors.  After 
removing  the  scapulae  by  means  of  bone  forceps  the  muscles  are  re- 
moved from  both  sides  of  the  vertebral  column. 

The  skull  is  then  grasped  firmly  in  a  pair  of  strong  bone  pincers 
(Paraboeuf 's),  and  the  upper  portion  is  removed  by  means  of  a  pair  of 
bone  forceps.  The  tops  of  the  vertebras  are  removed  in  like  manner, 
piece  by  piece,  exposing  thus,  the  entire  brain  and  spinal  cord.  Care 
must  be  taken  to  avoid  injury  to  the  brain  or  spinal  cord.  The  lower 
end  of  the  cord  is  then  held  by  forceps  and  all  connections  with  the 
vertebral  canal  are  cut.  The  cord  is  then  laid  back  in  the  canal. 
The  cord  is  used  for  the  preparation  of  Pasteur's  anti-rabic  vaccine. 
The  several  steps  taken  are  herewith  indicated. 

A  noose  of  sterile  silk  thread  is  slipped  over  the  end  of  the  cord 
and  drawn  tight.  The  cord  is  then  cut  about  6  cm.  from  the  end 
and  suspended  from  the  neck  of  a  sterile  bottle.  The  latter  contains 
some  solid  caustic  potash  or  soda  on  the  bottom.  It  is  plugged  with 
cotton  and  is  marked  C.  A  second  slip-knot  is  then  placed  over  the 
cut  end  of  the  cord  and  any  connections  of  the  latter  with  the  canal 
are  severed  and  finally  the  cord  is  cut,  leaving  a  piece  about  6  cm. 
long  attached  to  the  thread.  This  is  suspended  like  the  former  in  a 


504  BACTERIOLOGY. 

bottle  marked  B.  In  like  manner  the  upper  third  of  the  cord  is  tied 
with  a  silk  thread,  the  connections  cut  loose,  and  lastly  the  cord  is 
severed  below  the  pons.  This  section  of  the  cord  is  suspended  in  a 
bottle  marked  A. 

The  bottles  containing-  the  cords  are  set  aside  at  a  constant  tem- 
perature of  about  23°.  The  cords  thus  undergo  desiccation,  and,  as  a 
result,  the  virus  gradually  loses  its  virulence  and  becomes  attenuated. 
From  day  to  day,  a  portion  of  each  cord  is  cut  off  and  planted  in  bouil- 
lon. If  bacteria  develop  it  must  be  discarded.  Suspensions  in  steril- 
ized water  or  bouillon  of  the  desiccated  cords  are  employed  in  the  pre- 
ventive inoculations  against  rabies. 

The  brain  and  medulla  are  removed  and  placed  in  a  sterile  dish. 
A  portion  of  the  medulla  about  the  size  of  a  grain  of  wheat  is  placed 
in  a  sterilized  test-glass  (Pig.  43).  By  means  of  a  sterile  drawn-out 
tube  pipette,  a  few  drops  of  sterile  water  are  added  from  time  to  time, 
and  the  tissue  is  reduced  to  a  pulp  by  means  of  a  sterile  glass  rod. 
When  the  insoluble  matter  has  settled,  the  cloudy  suspension, 
amounting  to  about  3  c.c.  is  drawn  up  into  a  sterile  syringe.  The 
needle  is  passed  through  a  sterile  paper  and  any  air  present  is  care- 
fully expelled.  A  drop  or  two  of  this  material  can  now  be  injected 
into  another  animal.  The  remainder  of  the  material  can  be  used  to 
make  the  necessary  bacteriological  control  examinations.  In  suspected 
cases  the  medulla,  or  brain  should  be  placed  in  sterile  glycerin.  In 
this  condition  the  virus  retains  its  virulence  for  many  weeks.  The 
material  can  thus  be  transported  to  a  laboratory  and  examined  in  the 
manner  indicated. 


CHAPTER     XV. 

SPECIAL  METHODS  OF  WORK,  Continued. 
Serum  Agglutination. 

The  normal  serum  of  the  blood  of  various  animals  on 
contact  with  certain  motile  bacteria  (Sanarelli,  Eberth, 
Colon,  etc.)  will  cause  these  to  clump  together  or  gather 
into  masses.  At  the  same  time  the  individual  cells  lose 
their  motion  and  become  as  it  were  paralyzed.  This  phe- 
nomenon is  known  as  the  agglutination  reaction.  If  the  nor- 
mal serum  is  diluted  the  reaction  will  be  less  intense,  and 
frequently  will  cease  to  be  given  when  the  serum  is  diluted 
with  10  parts  of  water.  Occasionally,  a  normal  serum  will 
still  agglutinate  in  a  dilution  of  1 : 20  or  even  1 :  30. 

In  certain  diseases,  as  nT  typhoid  fever,  the  agglutina- 
ting substance  is  greatly  increased  in  amount.  The  reac- 
tion then  will  be  surely  given  with  the  Eberth  bacillus 
when  the  serum  is  diluted  1 : 30,  and  may  be  given  with  a 
dilution  of  1 :  50  or  even  with  greater  dilutions.  It  will  be 
seen,  therefore,  that  the  reaction  must  be  quantitative  in 
character  when  applied  to  the  blood  of  a  suspected  typhoid 
fever  case.  The  following  method  permits  an  accurate  es- 
timation of  the  agglutinating  power  of  a  serum. 

The  blood  is  obtained  by  puncture  of  the  finger  and  is 
allowed  to  clot.  The  clot  can  then  be  loosened  with  a  wire 
and  the  mixture  centrifugated.  The  clear  serum  is  used  for 
the  test.  An  abundance  of  blood,  and  hence  of  serum,  can 
be  obtained  from  a  vein  in  the  manner  described  on  p.  462. 
The  serum  and  the  diluting  flu;d  should  be  measured  by 
means  of  a  drawn-out  tube  pipette  (Fig.  61,  p.  457).  The 


506  BACTERIOLOGY. 

drawn-out  end  of  the  pipette  should  be  cut  square.  The 
dilution  of  the  serum  can  be  made  with  sterile  water,  phys- 
iological salt  solution,  or  with  bouillon. 

A  number  of  glass  cups  (Klotze)  are  placed  on  a  mount- 
ing- board  (p.  279).  By  means  of  the  pipette  these  receive  10, 
20,  30,  40,  and  50  drops,  respectively,  of  the  sterile  water. 
The  serum  is  then  drawn  up  into  the  same  pipette  and  one 
drop  of  it  is  placed  in  each  of  the  dishes.  The  dilutions 
1: 10,  1:  20,  1:  30,  1:  40,  1:  50  are  then  prepared.  The  liquids 
should  be  thoroughly  mixed.  This  can  be  done  expedi- 
tiously  and  thoroughly  by  means  of  the  pipette  used.  This 
should  be  placed  in  a  tumbler  of  water  and  the  inside 
washed  by  repeatedly  drawing  in  water.  The  pipette  thus 
rinsed  is  now  introduced  into  the  weakest  dilution  (1 :  50) 
and  the  liquid  is  drawn  up.  It  is  then  forced  out  and  this 
operation  repeated  two  or  three  times  will  bring  about  a 
thorough  mixing  of  the  serum  and  water.  The  pipette  is 
again  rinsed  in  clean  water  and  the  next  dilution  (1 : 40)  is 
mixed  in  the  same  manner.  In  this  way  each  dilution  is 
rendered  homogeneous. 

A  number  of  clean  cover-glasses  are  laid  on  the  board. 
A  drop  from  each  of  the  above  dilutions  is  placed  on  the 
corresponding  cover-glass.  Each  drop  is  then  inoculated 
with  a  minute  portion  of  an  agar  culture  of  the  Eberth  ba- 
cillus. The  culture  should  be  recent,  preferably  12  to  18 
hours  old.  A  concave  slide,  ringed  with  vaselin,  is  brought 
over  each  cover-glass  and  the  hanging-drops  (p.  143),  thus 
prepared,  are  ready  for  examination. 

The  specimens  should  be  examined  immediately,  and  at 
the  end  of  >^,  1  and  1^  hours.  If  agglutination  does  not 
occur  in  this  time  it  is  unnecessary  to  prolong  the  observa- 
tion. The  undiluted  serum  should  be  tested  at  the  same 
time  as  the  others.  Moreover,  one  or  more  control  hanging- 
drops  should  be  prepared  in  order  to  make  certain  that  the 
organism  does  not  exist  already  clumped  in  masses  in  the 
culture  tube.  In  case  the  1 :  50  dilution  gives  a  marked  ag- 


POISONOUS  FOODS.  507 

glutination,  the  test  should  be  repeated  with  higher  dilu- 
tions. Thus,  1  drop  of  1 : 10  added  to  10  drops  of  water 
will  give  1:100. 

Some  workers  prefer  to  make  the  dilutions  with  a 
young-  bouillon  culture  (6  to  8  hours  at  37°)  of  the  Eberth 
bacillus.  This  is  intended  to  obviate  the  possible  error  of 
mistaking  the  clumps  of  bacteria  as  occasionally  found  in 
agar  cultures  for  the  real  agglutinated  masses. 

The  reaction  is  applicable  to  dried  blood  and,  indeed,  this  is  per- 
haps the  most  convenient  way  of  sending-  the  material  for  an  examin- 
ation. The  blood  is  allowed  to  fall,  drop  by  drop,  on  different  portions 
of  a  piece  of  filter-paper.  When  the  blood  has  dried  perfectly  the 
paper  may  be  folded  and  mailed.  The  blood-stained  circles,  each  repre- 
senting one  drop  of  blood,  are  cut  out  and  placed  in  dishes  containing 
10,  20,  30,  etc.,  drops  of  water,  respectively.  The  solution  of  the  blood 
constituents  is  facilitated  by  crushing-  the  bit  of  paper  with  a  glass  rod. 
Hang-ing-drops  are  then  prepared  in  the  manner  indicated  above. 

According  to  Johnston  and  MacTaggart  pseudo-reactions  are 
characterized  by  rapid  clumping  without  the  corresponding  loss  of 
motion  which  is  so  characteristic  of  the  true  reaction.  After  a  few 
hours  these  clumps  tend  to  break  up.  These  pseudo-reactions  can  be 
avoided  by  using  an  attenuated  culture  which  is  transplanted  at  in- 
tervals of  about  a  month  and  grown  at  the  room  temperature.  Prom 
such  stock  cultures  a  bouillon  culture  is  made  and  developed  at  37° 
for  24  hours.  This  is  then  employed.  By  using  such  attenuated  cul- 
tures the  authors  do  not  consider  it  necessary  to  make  dilution  tests. 
A  large  drop  of  water  is  placed  on  the  blood  stain  and  allowed  to 
stand  for  a  minute  or  two.  A  loopful  of  the  solution  thus  obtained  is 
taken  from  the  top  of  the  drop  and  mixed  with  a  loopful  of  the 
bouillon  culture.  The  reaction  is  usually  given  in  this  method  in 
about  15  minutes. 

Poisonous  Foods'. 


The  examination  of  food  which  is  suspected  to  be  the 
cause  of  illness  is  frequently  demanded.  Before  the  devel- 
opment of  bacteriology  these  cases  of  poisoning  were 
usually  believed  to  be  due  to  metallic  poisons  which  were 
introduced,  accidentally  or  otherwise,  into  the  food.  The 


508  BACTERIOLOGY. 

possibility  of  the  presence  of  injurious  metals,  such  as  tin 
or  arsenic,  should  be  conceded  and  a  thorough  examination 
of  a  poisonous  food  should  involve  tests  for  the  recognition 
of  such  metals.  On  the  other  hand,  the  fact  that  a  gram 
or  two  of  cheese,  or  a  tea-spoonful  of  ice-cream,  at  -times, 
causes  pronounced  illness  would  indicate  that  the  poison 
cannot  belong  to  the  group  of  metals. 

In  the  examination  of  poisonous  meat  and  sausage  the 
possibility  of  the  presence  of  trichinae  should  be  clearly 
borne  in  mind.  A  bacteriological  examination  will  be  of 
no  value  unless  the  presence  of  these  parasites  is  definitely 
excluded.  Repeated  examinations  should  be  made,  and,  if 
possible,  these  should  be  controlled  by  the  examination  of 
tissue  known  to  contain  trichinae. 

Having  excluded  the  presence  of  metallic  poisons  and 
the  presence  of  animal  parasites,  as  trichinae,  then  the 
poison-producing  bacteria  that  may  be  present  should 
receive  attention.  Food-poisoning  from  bacteria  may 
originate  in  any  one  of  the  following  ways:  1. — The  food  is 
infected  and  the  poison  is  generated  exclusively  be- 
fore the  food  is  taken.  The  organism  in  this  case  may  be 
considered  as  a  true  saprophyte  endowed,  however,  with  the 
property  of  producing  highly  poisonous  substances.  Hence, 
the  intoxication  will  be  directly  proportional  to  the 
amount  of  poison  which  is  ready  made  in  the  food  at  the 
time  this  was  taken.  2. — The  infecting  organism  may 
begin  the  elaboration  of  its  poisonous  products  outside  of, 
and  continue  the  same  process  inside  the  body.  In  this 
case  the  organism  is  able  to  grow  in  the  intestines,  and  may 
even  penetrate  the  organs  and  tissues.  The  illness  has  the 
general  characteristics  of  an  acute  infectious  disease. 
3. — The  illness  may  not  result  in  the  production  of  poisons 
until  the  food  is  taken  into  the  body.  As  an  illustration, 
milk  or  water  infected  with  typhoid  or  cholera  bacteria 
may  give  rise  in  the  one  case  to  typhoid  fever,  and  in  the 
other  case  to  cholera. 


PURIFICATION  OF   LITMUS.  509 

A  proper  bacteriological  examination  must  take  into  account 
the  several  possibilities  just  mentioned.  Inasmuch  as  anaerobic  bac- 
teria may  be  the  cause  of  such  food  infection,  plates  should  also  be 
made  in  glucose  gelatin  and  in  glucose  agar  and  allowed  to  develop  in 
an  anaerobic  apparatus  (p.  314).  The  gelatin  plates  are,  of  course, 
developed  at  ordinary  room  temperature,  whereas  the  agar 
plates  should  be  grown  at  37°.  The  several  varieties  of  colonies 
should  be  transplanted  and  the  effects  of  the  pure  living  cultures,  and 
also  of  the  filtered  cultures,  should  be  studied  on  animals.  The  animal 
employed  should,  if  possible,  be  susceptible N  to  the  poisonous  food 
itself.  In  many  cases,  the  dog  and  cat  are  preferable  to  the  ordinary 
experimental  animals. 

At  the  time  the  plate  cultures  are  planted  bouillon  tubes  should 
be  inoculated  and  allowed  to  develop,  some  at  37°  and  some  at  ordinary 
room  temperature.  The  cultures,  thus  obtained,  can  be  tested  by 
subcutaneous  injection  into  guinea-pigs  or  rabbits.  In  case  of  the 
death  of  the  experimental  animal,  the  serous  fluid  in  the  large  cavi- 
ties and  especially  the  heart-blood  should  be  used  to  obtain  pure 
cultures  of  the  pathogenic  organism  which  is  present. 


Purification  of  Litmus. 

The  ordinary  litmus  cubes,  as  met  with  commercially, 
do  not  yield  a  pure  blue  solution  of  the  pigment.  This  is 
due  to  the  presence  of  various  impurities,  notably  of  a  red 
pigment.  A  pure  blue  litmus  solution  can  be  readily  pre- 
pared according  to  the  following  directions: 

25  g.  of  litmus  are  placed  in  a  beaker  and  250  c.c.  of 
distilled  water  are  added.  The  solution  is  then  heated  on 
a  boiling  water-bath  or  in  steam  for  about  30  minutes.  The 
deep  blue  liquid  is  then  decanted;  water  is  added  to  the 
residue  and  the  mixture  heated  as  before,  and  finally  the 
blue  solution  is  again  decanted.  This  washing  out  of 
the  residue  is  repeated  once  or  twice.  The  combined 
aqueous  extracts  are  allowed  to  settle  over  night  and  are 
then  filtered.  The  filtrate  is  concentrated  in  an  evaporat- 
ing dish,  over  a  flame,  to  less  than  one-half.  It  is  then 


510  BACTERIOLOGY. 

filtered  again  and  the  concentration  is  continued  till  about 
50  c.c.  of  liquid  are  left. 

The  concentrated  blue  liquid  is  then  poured  gradually, 
with  constant  stirring,  into  about  5  volumes  of  absolute 
alcohol.  The  blue  pigment  is  thrown  down  as  a  sticky 
precipitate,  whereas  the  red  color  remains  in  solution  in 
the  alcohol.  The  precipitate  is  allowed  to  settle  thorough- 
ly; the  alcoholic  liquid  is  then  decanted  as  much  as  possible 
or  it  is  removed  by  filtration. 

The  sticky  precipitate  is  then  dissolved  in  about  50  c.c. 
of  -water  and  the  blue  pigment  is  again  precipitated  by 
pouring  the  solution,  as  directed  above,  into  5  volumes  of 
strong  alcohol.  The  alcoholic  liquid  is  again  decanted  and 
is  finally  drained  off  as  much  as  possible. 

The  mass  now  contains  the  purified  blue  pigment, 
mixed  with  various  lime  salts.  Inasmuch  as  the  pigment 
may  not  possess  the  requisite  degree  of  sensitiveness  it 
is  well  to  make  it  as  sensitive  as  possible.  For  this  pur- 
pose, about  250  c.c.  of  water  should  be  added  to  the  pre- 
cipitate and  the  mixture  warmed  on  the  water-bath  till 
complete  solution  has  taken  place  place.  Then  dilute 
H2SO4  is  added,  drop  by  drop,  till  a  portion  of  the  liquid 
placed  in  a  tube  and  diluted  has  a  strong  red  color.  The 
liquid  is  then  boiled  to  expel  CO2  and  alcohol.  It  is  then 
neutralized  by  adding  saturated  baryta  water,  drop  by  drop, 
and  stirring  well.  This  addition  of  baryta  is  continued  till 
a  few  drops  of  the  solution,  placed  in  a  test-tube  and  diluted 
with  water,  show  a  clear  blue  liquid  without  a  tinge  of  red. 
If  a  drop  or  less  of  o  HC1  is  added  to  the  tube  it  will 
promptly  change  to  a  deep  red.  The  liquid  is  now  boiled 
for  a  few  minutes  and  set  aside  over  night.  The  precipitate 
tof  barium  and  calcium  salts  is  then  removed  by  filtration. 
The  purified,  concentrated  litmus  solution  is  then  placed  in 
Billings'  glass-stoppered  flasks  and  sterilized  by  steam. 
Whenever  it  is  desired  to  add  the  litmus  to  sterile  lactose 
agar,  glucose  agar,  glucose  gelatin,  milk,  etc.,  it  should  be 


TUBING  OP  MEDIA. 


511 


drawn  up  into  a  sterile  bulb  pipette  (Fig-.  61),  and  then 
transferred  direct  to  these  tubes.  The  addition  of  sterile 
litmus  solution  to  sterile  media  is  preferable  to  the  ordinary 
method  of  adding  the  litmus  to  the  medium  before  steril- 
ization. 

Tubing  of  Media. 


In  many  experiments,  as  in  the  disinfection  tests  pres- 
ently to  be  described,  it  is  desirable  to 
employ  measured  amounts  of  bouillon. 
This  can  be  done  expeditiously  by  means 
of  the  simple  apparatus  shown  in  Fig. 
72.  The  lower  end  of  a  100  c.c.  burette 
is  attached  to  a  small  glass  T-tube  which 
connects  with  the  reservoir  flask.  The 
burette  can  thus  be  readily  filled  and 
exact  amounts  can  be  measured  out  into 
the  tubes. 

For  rapid  tubing  of  gelatin,  bouillon, 
agar,  etc.,  where  it  is  not  essential  to 
employ  a  definite  volume  it  is  well  to 
employ  a  funnel  or  glass  globe  which  is 
connected  by  means  of  rubber  tubing  with 
a  drawn-out  glass  tube.  A  Mohr's  clamp 
is  attached  to  the  rubber  tubing  or  in  its 
stead  a  glass  stop-cock  may  be  used. 
The  perforation  in  the  latter  should  have 
a  diameter  of  about  3  mm.  Such  an  ap- 
paratus is  obtained  by  disconnecting  the  £.IG-  72.  Apparatus  for 

J  tubing  definite  quantities 

long  rubber  tube  at  a  in  Fig.   72.      This  ?ubh!gdidiscoFn0nrecTdiantar« 
simple  apparatus  can,  therefore,   be   em-(F'G>N>)' 
ployed  for  ordinary  tubing  of  media,  or  for  measuring  out 
definite  volumes. 


512 


BACTERIOLOGY. 


The  Sealing  and  Keeping  of  Cultures. 


The  method  of  preserving  cultures  or  specimens  of 
blood,  etc.,  in  drawn-out  bulb  tubes  has  been  touched  upon 
(p.  458).  In  this  connection  it  is  desirable  to  indicate  the 
methods  employed  in  sealing  the  ordinary  culture  tubes. 
Three  or  four  procedures  are  resorted  to.  The  tubes  may 
be  sealed  with  rubber  caps,  corks,  sealing-wax,  or  with 
paraffin.  Thin  sheets  of  rubber  may  also  be  used. 

In  either  case,  the  cotton  plug  should  be  cut  close  to  the 
end  of  the  tube.  It  should  then  be  drawn  out  slightly  and 
rolled  rapidly  in  the  flame  till  the  cotton  is  charred  a  trifle. 
The  cotton  is  then  pushed  into  the  tube  to  prevent  smold- 
ering. If  the  tubes  are  sealed  without  this  precaution, 
moulds  are  liable  to  develop  starting  from  the  cotton  plug. 


FIG.  73.    The  keeping  pf  cultures  in  black,  paper  boxes   (F.  G.  N.). 

The  corks  or  rubber  caps  should  be  immersed  in  mer- 
curic chloride  solution  (1-1000)  and  steamed  for  at  least  15 
minutes.  They  can  then  be  used  for  sealing  the  tubes. 
The  rubber  caps,  undoubtedly,  are  the  most  convenient  for 
this  purpose.  They  are,  however,  expensive  and  are  likely 
to  deteriorate  on  keeping.  The  corks  are  not  only  steril- 
ized by  the  exposure  to  steam,  but  they  are  also  softened. 
They  can  now  be  easily  inserted  into  the  tubes,  and  a  per- 
fectly tight  closure  can  be  obtained.  When  caps  or  corks 


THERMAL  DEATH-POINT.  513 

are  used  the  cotton  plug  should  be  pushed  down  into  the 
tube  (1-1.5  cm.)  by  means  of  sterile  forceps.  It  should  not 
touch  the  rubber  cap  or  cork. 

A  very  cheap  and  satisfactory  way  of  sealing-  tubes  is 
to  employ  sealing-wax.  The  cotton  plug  should  be  firm 
and  solid,  and  should  not  be  pushed  below  the  level  of  the 
mouth  of  the  tube.  The  heated  wax  should  be  first  applied 
to  the  edge  of  the  cooled  tube,  and  finally  the  center  of  the 
plug  should  be  covered. 

The  tube  cultures  are  usually  kept  in  tumblers  in  a 
darkened  case.  The  author  employs  for  this  purpose  black, 
jacketed  boxes  shown  in  Fig.  73.  The  box  is  6.5  cm.  wide, 
10  cm.  deep  and  18  cm.  high.  The  lid  is  9  cm.  high,  whereas 
the  inner  height  of  the  lower  portion  is  12.5  cm.  A  large 
number  of  boxes  can  thus  be  arranged  like  books  on  a  shelf. 
The  cultures  are  protected  perfectly  from  dust  and  light. 

Thermal   Death-point. 

\ 

As  indicated  heretofore,  the  vegetative  and  spore  form 
of  the  same  organism  differ  in  their  resistance  to  destruc- 
tion. Thus,  while  the  former  is  readily  destroyed  by  a  tem- 
perature of  60-70°,  the  latter  requires  an  exposure  to  steam 
for  some  minutes.  The  vegetating  forms  of  different  species 
of  bacteria  likewise  show  different  degrees  of  resistance. 
Some  are  killed  readily  at  55°  and  others  require  65  to  70°. 
Moreover,  it  should  be  borne  in  mind  that  several  varieties 
of  a  given  species  may  exist  no  two  of  which  necessarily  are 
equally  resistant.  Thus,  we  may  have  spores  of  anthrax 
which  will  be  destroyed  by  a  5  per  cent,  solution  of  carbolic 
acid  within  24  hours,  while  another  yield  of  spores  from  a 
different  variety  of  the  same  organism  may  resist  this  same 
disinfectant  for  50  days  or  longer. 

In  studying-  the  resistance  of  an  organism  it  is  essential  to  pre- 
pare as  nearly  a  homogeneous  suspension  as  possible.  That  is  to  say,. 


514  BACTERIOLOGY. 

each  germ  present  should  be  single  and  wholly  free  from  contact  with 
other  organisms.  The  massing"  of  bacteria  necessarily  affords  protec- 
tion to  the  cells  in  the  center  of  such  groups.  Because  of  this  protec- 
tion these  few  cells  may  survive,  while  all  the  others  may  be  destroyed. 
The  results  obtained  would  consequently  be  misleading.  This  error 
can  be  avoided  by  filtering  the  suspension  through  sterile  absorbent 
cotton  or  glass-wool  in  the  manner  presently  to  be  described. 

When  it  is  desired  to  test  the  resistance  of  the  vegeta- 
tive form  of  a  given  organism  special  care  must  be  taken  to 
exclude  the  presence  of  spores.  Many 
bacteria  like  those  of  typhoid  fever, 
cholera,  etc.,  do  not  form  spores  and. 
hence  such  precautions  are  not  observed 
when  testing  these  organisms.  The 
anthrax  bacillus,  however,  does  form 
spores,  and  hence  in  testing  the  resist- 
ance of  the  growing  bacillus  the  spore 
form  must  be  eliminated.  This  is  ac- 
complished by  making  so-called  homo- 
geneous cultures.  These  are  obtained 
by  making  transplantations  to  bouillon 
every  6  or  8  hours.  After  several  such 
transplantations  only  actively  growing 
bacteria  will  be  present. 

The  best  procedure  for  studying  the 
FIG.  74.  Filter  for  bacterial  action  of  moist  heat  on  bacteria  is  to 

suspensions.  .       '.\_. 

place  homogeneous  suspensions  in  thin, 
straight  capillary  tubes.  It  is  essential  that  the  walls 
of  the  capillary  tube  shall  be  as  thin  as  possible.  They 
should  not  have  a  bulb  of  thick  glass.  When  such  sealed 
capillary  tubes  are  immersed  they  rapidly  acquire  the  tem- 
perature of  the  surrounding  liquid. 

Preparation  of  ^bacterial  suspension. — As  a  rule,  it  is  best  to  em- 
ploy young  cultures  on  agar.  Sterile  bouillon  is  introduced  into  the 
test-tube  by  means  of  a  drawn-out  pipette  (Fig.  61  e]  and  the  growth 
is  thoroughly  rubbed  up.  The  suspension  is  now  drawn  up  into  the 
pipette  and  is  transferred  to  a  sterile  filter  (Fig.  74),  The  filter-tube 


THERMAL  DEATH-POINT.  515 

can  be  readily  made  by  drawing-  out  a  test-tube.  A  layer  of  absorbent 
cotton  should  be  placed  on  the  bottom  of  the  tube  and  then  some 
glass-wool  which  acts  as  a  weight.  The  filter  thus  prepared  is  plugged 
at  both  ends  and  sterilized.  The  filtered  suspension  should  be  exam- 
ined under  the  microscope,  and,  if  aggregations  of  bacteria  are  still 
present,  it  should  be  again  filtered.  The  filtrate  may  be  received  in  a 
large  Esmarch  dish,  or  in  a  sterile  beaker  which  is  covered  with 
cotton  or  paper. 

The  suspension  is  drawn  up  into  a  sterile  capillary 
pipette  which  is  then  sealed  below  and  above  the  liquid 
(Fig.  62  c  x,  p.  459).  The  liquid  is  now  contained  in  a  capil- 
lary tube  about  10  cm.  long  and  1-2  mm.  in  diameter.  A 
sufficient  number  of  these  sealed  .capillary  tubes  should  be 
prepared  (see  p.  516)  to  meet  the  requirements  of  the  ex- 
periment in  view.  They  should  be  immersed  in  a  test-tube 
containing  mercuric  chloride  in  order  to  disinfect  the 
exterior.  These  tubes  are  used  to  test  the  action  of  moist 
heat.  The  same  suspension  is  used  for  the  preparation  of 
silk  threads,  muslin  squares  and  cover-glass  specimens 
{p.  517). 

MOIST   HEAT. 

The  ordinary  Hofmann  iron  water-bath  will  answer 
very  well  for  these  tests.  It  is  filled  with  water  and  provided 
with  a  thermometer  and  a  thermo-regulator  which  should 
be  suspended  so  as  not  to  touch  the  bottom  or  the  sides 
(Pig.  75).  The  temperature  of  the  water  can  thus  be  kept 
at  any  desired  point.  The  water-bath  of  Roux,  shown  in 
Mg.  64,  is  very  useful  for  these  experiments.  The  capillary 
tubes  should  be  placed  on  cross  wires  which  are  stretched 
over  a  syringe  holder  (Fig.  41  c,  p.  263),  and  immersed  in  the 
water.  At  the  end  of  stated  intervals,  the  holder  is  raised 
and  a  capillary  tube  is  removed.  The  latter  should  be 
placed  at  once  in  a  tube  of  cold  water,  to  prevent  the  fur- 
ther action  of  heat.  Each  of  the  remaining  capillary  tubes, 
in  like  manner,  is  removed  and  placed  in  water  at  the  end 
of  the  respective  period  of  exposure. 


516 


BACTERIOLOGY. 


The  contents  of  each  of  the  capillary  tubes  are  then  in- 
oculated into  bouillon  which  is  set  aside  at  37°  to  allow  the 
growth,  if  any,  to  develop.  The  contents  are  removed 
as  follows:  The  tube  is  first  wiped  dry  and  then  one 

end  is  slig-htly  scratched  with 
a  file.  The  end  is  then  removed 
and  .the  opening-  of  the  tube  is 
sterilized  by  touching'  it  2  or  3 
times  to  a  flame.  The  tube  is 
held  in  a  broad-pointed  pair  of 
forceps,  and,  when  cool,  the  cut 
end  is  inserted  into  the  culture 
tube  while  the  closed  end  is 
slowly  brought  into  contact  with 
a  flame.  The  vapor  thus  pro- 
duced promptly  expels  the  liquid 
from  the  capil- 
lary. 

The  student  should 
make  suspensions 
and  test  one  or  more 
of  the  following-  or- 
ganisms, according- 
to  the  directions 
given  above.  Chol- 
era vibrio,  Typhoid 
FIG.  75.  Determination  of  the  thermal  death-point  (F,  G.  N.)-  bacillus,  Anthrax 

bacillus  (homogeneous 
culture),  Anthrax  spores  (p.  291)  and  Streptococcus  pyog-enes. 

Action  of  moist  heat  at  58°.— The  capillary  tubes  should  be  with- 
drawn at  the  end  of  5,  10,  15,  30  and  60  minutes'  exposure. 

Action  of  moist  heat  at  70°.—  The  capillary  tubes  are  withdrawn  at 
the  end  of  1,  3,  5  and  10  minutes.  In  addition  to  this  the  anthrax 
spore  tubes  should  be  exposed  for  15,  30  and  60  minutes. 

Action  of  moist  heat  at  100°. ~ The  capillary  tubes  are  withdrawn  at 
the  end  of  1,  2  and  3  minutes.  Additional  anthrax  spore  tubes  are  ex- 
posed for  5,  10  and  15  minutes. 


THERMAL    DEATH-POINT.  517 

DRY   HEAT. 

The  ordinary  dry  heat  sterilizer  is  employed  in  study- 
ing the  action  of  this  form  of  heat  on  bacteria.  The  bulb 
of  the  thermometer  should  be  placed  so  as  to  be  on  a  level 
with  the  objects  exposed.  It  should  not,  however,  rest  on 
a  metal  surface.  A  thermometer  is  used  to  maintain  a  con- 
stant temperature. 

The  suspensions  prepared  as  above  cannot  obviously  be 
employed  as  such.  It  is  customary  to  soak,  in  these  sus- 
pensions, bits  of  sterile  silk  threads  or  small  squares  of 
muslin.  These  are  then  allowed  to  dry  before  they  are 
used.  Sterile  cover-glasses  are  also  smeared  on  one  side 
with  the  suspended  bacteria,  and  when  dry  they  can  be  used 
in  a  similar  manner. 

The  silk  threads  are  prepared  by  cutting  up  some  of 
the  silk  in  lengths  of  about  1.5  cm.  These  should  be  placed 
in  a  plugged  test-tube  and  sterilized  in  the  dry-heat  oven. 
At  the  same  time  some  muslin  is  cut  up  into  squares  of 
about  1  cm.  on  a  side.  These  are  sterilized  in  a  Petri  dish. 
Thoroughly  clean  cover-glasses  (p.  140),  18  to  20mm.  square 
are  cut  into  halves  by  means  of  a  ruler  and  diamond.  The 
oblong  slips  of  glass  are  likewise  placed  in  a  Petri  dish  and 
sterilized. 

The  bacterial  suspension  prepared  as  above  (p.  514)  is 
transferred  to  a  sterile  wide  Esmarch  dish  and  the  proper 
number  of  silk  threads  and  muslin  squares  are  then  added 
and  are  allowed  to  soak  thoroughly.  One  by  one,  they  are 
then  picked  up  by  means  of  sterile  forceps,  and  arranged  in 
rows  in  a  sterile  Petri  dish.  A  corresponding  number  of 
the  sterile  cover-glasses  are  likewise  placed  in  the  Petri 
dish  and  each  one  is  covered  with  a  large  loopful  of  the  sus- 
pension. This  is  spread  over  as  much  of  the  surface  as  pos- 
sible and  care  is  taken  that  the  liquid  does  not  run  over  the 
edge  to  the  under  side  of  the  cover-glass.  The  silk-threads, 
muslin  squares  and  cover-glasses  are  then  allowed  to  dry 


518  BACTERIOLOGY. 

at  the  ordinary  temperature.  The  drying-  may  be  hastened 
by  placing1  the  dishes,  slightly  uncovered,  in  an  incubator 
at  37°  for  three  hours. 

When  the  temperature  of  the  oven  has  reached  the  de- 
sired point,  the  proper  number  of  silk  threads,  muslin 
squares  or  cover-glasses  are  placed  in  a  sterile  Petri  dish 
which  is  then  set  within  the  oven  on  a  level  with  the  bulb 
of  the  thermometer.  At  the  end  of  stated  intervals  a  silk 
Thread,  muslin  square  or  cover-glass,  is  taken  out  as  rapidly 
as  possible,  by  means  of  sterile  forceps,  and  is  transferred 
to  a  tube  of  nutrient  bouillon.  This  is  labelled  at  once,  and, 
eventually  the  entire  set  is  placed  in  the  incubator. 

With  infected  silk  threads,  prepared  as  above,  the  student  should 
test  the  action  of  dry-heat  on  the  several  organisms  already  studied. 
The  results  obtained  in  this,  and  in  all  other  laboratory  work,  should 
be  arranged  in  tabular  form  to  facilitate  comparison  and  review  of 
results. 

Action  of  dry  heat  at  70°.—  The  silk  threads  should  be  withdrawn  at 
the  end  of  15,  30,  45  and  60  minutes'  exposure. 

Action  of  dry  heat  at  100°.— The  specimens  should  be  withdrawn  at 
intervals  as  just  given. 

Action  of  dry  heat  at  120°.— The  specimens  should  be  withdrawn  at 
same  intervals  as  above. 

Action  of  dry  heat  at  150°.—  The  specimens  should  be  withdrawn  at 
intervals  of  5,  10,  15,  30,  45  and  60  minutes. 


Testing  of   Disinfectants. 

In  studying  the  action  of  physical  and  chemical  ag-ents 
on  bacteria  it  is  necessary  to  rigidly  adhere  to  certain  re- 
quirements without  which  the  results  would  be  of  little 
value,  if  not  wholly  contradictory.  The  conditions  which 
underly  the  testing-  of  disinfectants  may  be  summed  up  as 
follows : 


TESTING  OF  DISINFECTANTS.  519 

1. — Variable  resistance  of  spores  and  of  the  vegetating  forms 
of  one  and  the  same  organism.  It  has  been  shown  in  recent 
years  that  considerable  variation  may  exist  in  the  resistance 
which  an  organism  possesses  to  destruction.  Thus,  while 
there  are  some  spores  of  anthrax  which  are  readily  de- 
stroyed by  steam-heat  (100°)  others  have  been  known  to 
.withstand  this  temperature  for  10-12  minutes.  Again,  it 
was  formerly  stated  that  anthrax  spores  were  destroyed  by 
5  per  cent,  carbolic  acid  in  two  days  but  the  researches  of 
Fraenkel  have  shown  that  spores  of  anthrax  may  be  had 
which  are  not  destroyed  by  an  exposure  of  30  to  40  days  or 
even  longer.  In  view  of  these  facts  several  standards  have 
been  proposed.  Thus,  Fraenkel  designates  anthrax  spores 
which  are  destroyed  by  5  per  cent,  carbolic  in  less  than 
10  days  as  feebly  resistant;  in  10  to  20  days  as  of  aver- 
age resistance;  in  20  to  30  days  as  very  resistant;  in  30  to  40 
days  as  extremely  resistant.  Geppert's  standard  anthrax 
spores  are  those  which  are  infectious  after  boiling  for  one 
minute  1  c.c.  of  a  spore  suspension  which  is  added  to  30  c.c. 
of  boiling  water.  Esmarch  has  suggested  as  a  standard, 
anthrax  spores  which  when  fixed  on  silk  threads  resist 
steam-heat  of  100°  for  10  minutes. 

2. — The  influence  of  the  medium  in  which  the  organism  is 
tested.  Thus,  it  has  been  shown  that  to  destroy  anthrax 
spores  in  bouillon  it  requires  20  times  as  much  mercuric 
chloride  (1-1000)  than  when  they  are  suspended  in  water; 
and,  250  times  as  much  are  necessary  when  they  are  dis- 
tributed in  blood- serum. 

3. — The  temperature  at  which  the  disinfection  is  made. 
The  higher  the  temperature  at  which  the  experiments  are 
made  the  more  rapid  and  energetic  will  be  the  action  of  the 
disinfectant.  Cholera  bacteria  are  not  destroyed  by  mer- 
curic chloride  (1-1000)  in  one  hour  at  -3°,  whereas  at  36° 
they  are  killed  in  a  few  minutes. 


520  BACTERIOLOGY. 

4. — Immediate  and  thorough  contact  with  the  disinfectant 
of  all  the  organisms  present.  This  can  be  done  only 
with  bacterial  suspensions  in  which  each  organism  is 
entirely  free  and  separate  from  others.  To  obtain  such  a 
suspension,  it  is  necessary  to  filter  through  glass-wool 
or  absorbent  cotton  and  then  to  agitate  the  liquid  thorough- 
ly, at  a  temperature  of  about  37°,  until  microscopical  exam- 
ination shows  no  aggregations  of  bacteria.  Silk  threads 
which  have  been  soaked  in  bacterial  suspensions  and  then 
dried  are  open  to  the  objection  that,  on  treatment  with  the 
disinfectant,  the  organisms  are  unequally  exposed  and  some 
may  even  be  protected  by  their  position.  The  same  objec- 
tion, to  a  less  degree,  applies  to  cover-glasses  on  which  a 
thin  film  of  the  suspension  has  been  deposited. 

5. — The  number  of  bacteria  in  a  given  experiment.  It 
can  be  shown  readily  that  the  greater  the  number  of 
bacteria  present  the  more  slowly  will  disinfection  take 
place.  In  order,  therefore,  that  the  results  may  be  com- 
parable, approximately  the  same  number  of  organisms 
should  be  present  in  each  experiment.  This  is  readily  as- 
certained by  diluting  a  small  portion  of  the  bacterial  sus- 
pension with  1000  parts  of  sterilized  water  and  then 
making  a  gelatin  plate  with  one  drop  of  this  dilution. 

A  more  rapid  procedure  is  to  dilute  2  or  3  drops  of  the 
suspension  to  100  c.c.  Freshly  distilled  water  containing 
1  or  2  per  cent,  of  formaldehyde  should  be  used  for  this 
purpose.  The  suspension  is  transferred  to  a  Thoma-Zeiss 
counter  and  the  number  of  bacteria  on  the  ruled  square 
counted.  Each  of  the  small  squares  represents  WTJIT  cu.  mm. 
and,  since  there  are  400  of  these,  the  total  square  corre- 
sponds to  iV  cu.  mm.  Hence,  the  number  of  bacteria  found 
under  this  square  multiplied  by  10,000  will  give  the  number 
present  in  1  c.c.  of  the  diluted  suspension.  It  is  well  to 
allow  20  or  30  minutes  for  the  bacteria  to  settle  before 
counting. 


TESTING  OF  DISINFECTANTS.  521 

6. — The  amount  of  disinfectant  which  is  carried  over  to 
each  sub-culture.  Thus,  when  the  disinfectant  is  applied 
to  the  bacterial  suspension  and,  at  the  end  of  stated 
intervals,  transfers  of  1-3  loopsful  of  the  mixture  are  made 
to  sterilized  nutrient  media,  a  sufficient  amount  of  the  dis- 
infectant may  be  carried  over  to  prevent  the  growth  of  the 
organism  although  it  may  still  possess  vitality.  This  has 
been  a  most  serious  source  of  error  in  the  past.  The  error 
is  more  marked,  the  greater  the  antiseptic  power  of  the 
disinfectant.  It  is,  of  course,  less  marked  where  the  sub- 
stance has  weak  antiseptic  properties,  and,  where  the 
transplantation  occurs  into  relatively  large  amounts  of  the 
nutrient  medium  (10  to  15  c.c.).  It  must  -be  remembered 
that  in  all  cases  the  first  action  of  a  disinfectant  is  to  at- 
tenuate the  organism,  and,  that  when  the  latter  is  in  this 
condition,  a  much  smaller  amount  of  the  disinfectant  will 
act  as  an  antiseptic  and  prevent  growth.  This  has  been 
especially  shown  to  be  the  case  with  reference  to  the  action 
of  mercuric  chloride  on  anthrax  spores.  /  Formerly,  it  was 
supposed  that  these  were  killed  by  this  substance  in  a 
strength  of  1-1000  in  one  minute.  If  the  mercury  which  is 
held  fast  by  the  silk  thread,  and  which  cannot  be  removed 
by  mere  washing,  is  rendered  inert  by  the  action  of 
hydrogen  sulphide  it  can  be  shown  that  the  organism  is 
alive  and  infectious  even  after  an  exposure  of  4  hours.  It 
may  even  possess  vitality  after  an  exposure  of  24  hours. 
The  first  action  of  the  disinfectant  is  to  attenuate  the  organ- 
ism, the  growth  of  which  is  then  prevented  by  mere  traces 
of  mercury.  One  part  in  two  million  according  to  Geppert 
suffices  to  produce  this  result. 

When  making  transplantations  in  the  subsequent 
work  on  disinfectants,  the  platinum  wire  should  be  provided 
with  a  large  loop  having  about  2  mm.  clear  diameter.  The 
droplet  of  mercuric  chloride  (1-1000)  adhering  to  this  loop 
weighs  about  10  mg.  Hence,  when  transplanted  to  10  c.c. 
of  bouillon  the  latter  will  contain  mercuric  chloride  in  the 


522  BACTERIOLOGY. 

proportion  of  1  to  1,000,000.  It  is  evident,  therefore,  that 
in  such  work  only  one  loopful  should  be  carried  over  into 
the  bouillon,  the  volume  of  which  should  not  be  less  than 
10  c.c. 

7. — Observation  of  the  sub-cultures  over  a  considerable 
length  of  time.  The  failure  of  tubes  to  develop  within  24 
hours  is  not  a  positive  indication  that  the  organism  has 
been  destroyed  by  the  disinfectant.  In  the  attenuated  con- 
dition the  organism  will  grow  more  slowly  than  it  would  if 
it  were  normal  and  in  possession  of  full  vitality.  More- 
over, as  stated  already,  traces  of  the  disinfectant  which 
are  carried  over  in  the  experiment  will  still  further  tend  to 
retard  the  growth.  For  these  reasons  the  tubes  should  be 
kept  under  observation  for  at  least  one  week  before  definite 
conclusions  can  be  drawn. 

8. — Temperature  at  which  the  sub-cultures  are  kept.  The 
organism  which  has  been  exposed  to  the  action  of  the  dis- 
infectant should  be  placed  under  conditions  which  are  the 
most  favorable  to  its  growth.  That  is  to  say,  tlie  best  nu- 
trient medium  and  the  most  suitable  temperature  should  be 
furnished.  Transplantations  made  into  gelatin  and  kept  at 
ordinary  room  temperature  frequently  fail  to  grow  while 
parallel  bouillon  and  agar  cultures,  placed  in  the  incubator, 
develop.  It  is,  therefore,  desirable  to  make  the  transplan- 
tation to  the  surface  of  inclined  agar  tubes  or  into  bouillon 
and  to  keep  the  tubes  under  observation  at  a  temperature 
of  about  37°  for  a  week  or  more. 

9. — Negative  experiments  with  animals  inoculated  with  or- 
ganisms exposed  to  heat,  or  to  the  action  of  chemicals  prove 
but  little.  The  organism  may  be  dead,  or  it  may  have  be- 
come attenuated  and  is  therefore  without  action.  In  the 
latter  case  it  may  still  grow  on  artificial  media.  Thus,  an- 
thrax spores,  which  are  exposed  to  the  boiling  temperature 


TESTING  OF  DISINFECTANTS.  523 

for  2  minutes  no  longer  kill  guinea-pigs,  but  nevertheless 
they  can  grow  in  tubes,  even  after  5  minutes'  exposure.  On 
the  other  hand,  positive  results  may  be  obtained  by  inocu 
lating  white  mice  or  guinea-pigs  with  the  mixture  of  bac- 
teria and  disinfectant;  whereas,  the  same  material  trans- 
planted to  a  nutrient  medium  may  fail  to  grow  owing  to  the 
antiseptic  power  of  the  disinfectant  which  is  carried  over. 

10. — Control  experiments. — A  dozen  or  two  of  the  uninocu- 
lated  bouillon  tubes  should  be  placed  in  the  incubator,  to- 
gether with  the  sub-cultures  proper,  in  order  to  eliminate 
possible  error.  Moreover,  as  an  additional  control,  at  least 
one  tube  of  bouillon  should  be  inoculated  with  the  original 
untreated  culture,  cover-glass  or  silk -thread. 

Frequently,  the  sub-cultures  yield  doubtful  results. 
The  bouillon  in  that  case  is  slightly  clouded  and  the  growth 
itself  is  rather  uncertain.  In  this  case,  the  tubes  should  be 
returned  to,  and  kept  in  the  incubator  for  about  a  week. 
Or,  one  or  two  loopsful  should  be  transplanted  to  freshly 
inclined  agar. 

If  the  growth  that  develops  shows  the  slightest  varia- 
tion from  the  normal  one,  it  should  be  examined  under  the 
microscope  in  order  to  exclude  contaminations. 

The  action  of  a  disinfectant  on  bacteria  may  be  studied 
by  bringing  it  into  contact  with  infected  silk-threads,  mus- 
lin squares,  cover-glasses,  or  by  mixing  direct  with  the  bac- 
terial suspension.  The  preparations  of  these  specimens 
has  been  given  on  p.  517. 

Silk-threads. — This  method  was  introduced  by  Koch  and  has  been 
used  extensively.  Silk,  linen  or  cotton  threads  may  be  used.  They 
are  cut  up  into  convenient  lengths,  placed  in  a  plugged  tube,  steril- 
ized and  kept  for  future  use  (p.  517). 

To  ascertain  the  disinfecting-  action  of  a  solution,  a  dried  thread, 
impregnated  with  the  bacteria  to  be  tested,  is  immersed  in  it  for  a 
given  length  of  time,  as  for  instance,  2  minutes.  It  is  then  removed 
with  sterilized  forceps  and  gently  washed  in  sterilized  water  or  in  al- 


-524  BACTERIOLOGY. 

cohol.  Finally,  it  is  transferred  to  a  tube  of  nutrient  bouillon  (10  c.c.) 
and  then  set  aside  in  the  incubator  for  a  week  or  more.  Similar  tests 
with  exposures  of  5,  10,  30  and  60  minutes  are  made. 

The  objections  to  this  method  are  threefold  and  have  already 
been  incidentally  mentioned.  In  the  first  place,  the  bacteria  on  the 
thread  may  not  be  evenly  exposed  to  the  action  of  the  disinfectant; 
secondly,  the  disinfectant  itself  may  be  transferred  to  the  nutrient 
medium;  and  lastly,  a  small  amount  of  the  disinfectant  may  remain 
in  chemical  combination  with  the  silk  thread  and  by  its  presence  in- 
hibit the  development  of  the  already  attenuated  organism.  The  at- 
tempt is  made  to  obviate  the  second  objection  by  washing*  the  threads. 
This  may  be  successful  in  some  cases,  but  in  others  it  fails.  The  third 
objection  is  especially  true  of  mercuric  chloride  which  is  apparently 
held  fast  by  the  fiber  and  can  only  be  removed  by  the  action  of  hydro- 
gen sulphide  (Geppert). 

2. — Muslin  squares. — The  preparation  of  these  specimens  is  given 
on  p.  517.  They  are  used  chiefly  as  test-objects  in  experiments  on  the 
disinfection  of  rooms. 

3. — Cover-glasses.  This  method  was  introduced  by  Geppert  and 
has  been  used  by  Spirig  and  others.  To  test  a  disinfectant  a  dry 
•cover-glass,  streaked  with  the  organism  in  the  manner  described  on 
p.  517,  is  immersed  in  it  for  a  given  length  of  time  as  in  the  case  of 
the  silk  threads.  It  is  then  removed  with  sterilized  forceps  and 
washed  in  a  large  volume  of  sterilized  water  for  about  i-i  hour.  The 
cover-glass  is  then  placed  in  sterilized  bouillon  which  is  set  aside  in 
the  incubator. 

The  advantages  of  this  method  are  (1)  that  a  thin  film  of  evenly 
spread  bacteria  is  employed;  and,  (2)  that  the  cover-glass  does  not 
unite  with  the  disinfectant,  as  is  the  case  with  the  silk  threads.  It  is 
open  to  the  objection,  which  holds  true  also  for  the  silk  threads,  that 
the  process  of  desiccation  tends  to  lower  the  vitality  of  the  organ- 
ism. Furthermore,  it  may  be  urged  that  the  disinfectant  has  not  free 
access  to  all  sides  of  the  bacteria. 

4. — Bacterial  suspensions. — This  method  in  some  of  its  modifica- 
tions is  the  one  which  is  commonly  employed,  and,  if  used  with  proper 
precautions,  it  will  yield  perfectly  reliable  results.  The  first  essen- 
tial is  to  secure  a  suitable  suspension  of  the  organism  to  be  tested. 
Directions  for  doing  this  are  given  on  p.  514. 

In  general  the  procedure  consists  in  adding  to  a  given  volume  of 
the  suspension  an  equal  volume  of  the  disinfectant  of  double  the 


TESTING  OF  DISINFECTANTS.  525 

strength  to  be  tested.  At  stated  intervals  (1,  2,  5,  10  minutes,  etc.) 
one  large  loopful  of  the  mixture  is  transferred  to  nutrient  bouillon 
(10  c.c.),  or  to  agar  and  these  tubes  are  then  set  aside  in  the  incubator. 
The  method  as  given  is  open  to  the  objection  that  an  apprecia 
ble  amount  of  the  disinfectant  is  transferred  each  time  to  the  cul- 
ture tubes  and  that  it  may  prevent  growth.  This  is  specially  true 
with  substances  which  possess  marked  antiseptic  properties,  as  mer- 
curic chloride.  Whenever  possible,  the  disinfectant  should  be  ren- 
dered inert.  Thus,  traces  of  mercuric  chloride  can  be  removed  by 
precipitation  with  hydrogen  sulphide.  With  other  substances  the 
error  is  not  so  marked  and  is  partly  counterbalanced  by  keeping  the 
tubes  in  the  incubator  for  many  days.  The  important  point  is  to  in- 
oculate into  a  large  volume  of  bouillon,  not  less  than  10  c.c.,  and  when 
this  is  done  the  results  with  mercury  disinfectants  are  as  good,  if  not 
better,  then  when  hydrogen  sulphide  is  used. 

Laboratory  work. — The  student  will  test  the  action  of  mercuric 
chloride  (1-1000),  carbolic  acid  (5  per  cent.),  lysol  (5  per  cent.)  and  for- 
maldehyde (5  per  cent.)  on  the  following  organisms:  Typhoid  bacillus, 
Staphylococcus  pyogenes  aureus,  and  Anthrax  spores.  The  results 
are  to  be  tabulated. 

Action  of  mercuric  chloride. — A  solution  of  mercuric  chloride  (1-500) 
in  distilled  water  is  prepared.  It  is  well  to  steam  for  a  few  minutes 
all  freshly  prepared  disinfectants  in  order  to  insure  freedom  from 
error. 

a. — By  means  of  a  sterile  pipette  10  c.c.  of  this  disinfectant  solu- 
tion are  placed  in  a  small,  sterile  Erlenmeyer  flask  or  in  an  Esmarch 
dish,  and  an  equal  volume  of  the  bacterial  suspension  (p.  514)  is  added, 
likewise  by  means  of  a  sterile  pipette.  The  liquid  is  mixed  at  once.  At 
intervals  of  5,  10, 15,  30,  60  and  120  minutes,  a  large  loopful  of  the  mix- 
ture is  transplanted  to  at  least  10  c.c.  of  sterile  bouillon.  The  loop 
used  should  have  a  clear  diameter  of  2  mm.  The  set  of  tubes,  properly 
labeled,  are  then  placed  in  the  incubator. 

b. — In  the  above  method  the  injurious  effects  of  the  mercury  car- 
ried over  in  the  transplantation  are  largely  counteracted  by  the 
large  volume  of  bouillon  employed.  The  effects  of  the  mercury  can 
be  almost  wholly  done  away  with  by  passing  HaS  through  the  suspen- 
sion. In  this  case,  however,  care  must  be  taken  to  avoid  two  possible 
errors.  As  a  result  of  the  passage  of  the  gas,  HC1  is  liberated  and, 
unless  it  is  promptly  neutralized,  it  may  affect  the  test-organism  .- 
Again,  the  precipitate  of  mercury  sulphide  tends  to  drag  down  the 


526  BACTERIOLOGY. 

suspended  bacteria,  and,  as  a  result,  the  liquid  may  contain  few  or 
none.  A  loopful  of  the  liquid,  transplanted  to  bouillon,  may  there- 
fore give  no  growth,  although  living-  organisms  may  be  present  in  the 
tube.  Constant  results  in  this  method  can  only  be  obtained  by  trans- 
planting several  larg-e  drops  by  means  of  a  drawn-out  tube  pipette. 

The  Liborius  tube  is  admirably  adapted  for  testing  by  this 
method.  A  small  amount  of  dry  Na2CO3  on  the  point  of  a  knife 
(about  25  mg.)  is  placed  in  each  tube.  These  are  then  sterilized  in 
the  dry-heat  sterilizer. 

To  20  c.c.  of  the  bacterial  suspension  an  equal  volume  of  the 
mercury  solution  is  added  observing  the  same  precautions  as  under  a. 
At  the  end  of  5,  10,  15,  30,  60  and  120  minutes  about  2.5 c.c.  of  the  mix- 
ture are  transferred  to  a  Liborius  tube  which  is  connected  at  once 
with  a  H2S  generator.  The  gas  should  be  passed  for  1  or  2  minutes, 
and  the  tube  is  then  set  aside  for  the  mercury  precipitate  to  subside. 
When  this  has  taken  place  2-3  drops  of  the  clear  liquid  are  trans- 
ferred by  means  of  a  sterile  drawn-out  pipette  (p.  457)  to  a  tube  of 
nutrient  bouillon,  which  is  eventually  set  aside  in  the  incubator. 
Obviously,  the  same  pipette  can  be  used  in  transferring-  the  several 
portions  of  the  original  mixture  to  the  Liborius  tubes,  but  a  separate 
pipette  must  be  used  for  each  inoculation  made  from  the  latter. 

When  the  mixture  undergoing  examination  contains  a  very 
small  amount  of  mercury  (1-5000),  or  when  gelatin  or  soap  is  present 
the  mercury  sulphide  will  not  precipitate  but  will  remain  in  solution. 
In  such  cases,  it  can  be  thrown  out  of  solution  by  adding  an  equal  vol- 
ume of  sterile  saturated  NaCl  solution  to  the  mixture  in  the  Liborius 
tube  before  the  H2S  is  passed. 

Action  of  carbolic  acid. — A  10  per  cent,  solution  of  "carbolic  acid  is 
first  prepared,  by  the  aid  of  gentle  heat.  5  c.c.  of  the  bacterial  sus- 
pension are  placed  in  a  sterile  test-tube,  Esmarch  dish  or  small  Erlen- 
meyer  flask,  and  an  equal  volume  of  the  slightly  warmed,  perfectly 
clear,  10  per  cent,  solution  of  carbolic  acid  is  added.  The  liquid  is  at 
once  thoroug-hly  mixed  and  cooled.  At  intervals  of  1,  3,  5,  10,  15,  and 
30  minutes  a  loopful  of  the  mixture  is  transferred  to  a  tube  of  bouil- 
lon which  is  labelled  and  eventually  placed  in  the  incubator. 

In  the  case  of  anthrax  spores  it  will  be  well  to  inoculate  bouillon 
tubes  with  the  mixture  at  the  end  of  1,  3,  6,  24,  and  48  hours.  The 
liquid  should  be  thoroug-hly  mixed  just  before  making  each  trans- 
plantation. 

Action  of  lysol.—The  mixture  of  disinfectant  and  suspension  is 
made  according  to  the  directions  just  given.  The  tests  are  likewise 
carried  out  in  the  same  way. 


TESTING   OF  ANTISEPTICS.  527 

Action  of  formaldehyde. — The  commercial  formaldehyde  contains 
approximately  40  per  cent,  of  the  active  constituent.  The  10  per 
cent,  solution  is  prepared  by  adding-  10  c.c.  of  formalin  to  30  c.c.  of 
sterile  water.  5  c.c.  of  this  solution  are  placed  in  a  sterile  Esmarch 
dish  or  test-tube  and  an  equal  volume  of  the  suspension  is  then  added. 
At  intervals  of  1,  3,  5, 10, 15,  30  and  60  minutes  a  loopful  of  the  mixture 
is  transplanted  to  bouillon. 

In  the  above  method  of  testing-  no  account  is  taken  of  the  numer- 
ical decrease  of  the  org-anisms  present.  This,  however,  can  readily  be 
done  by  making-  ag-ar  Petri  dishes  at  the  intervals  given  above.  The 
plates  are  placed  in  the  incubator  and  the  colonies  that  develop  can 
then  be  counted. 


Testing  of  Antiseptics. 

The  first  action  of  a  chemical  substance,  when  added  to 
a  recently  inoculated  culture  medium,  is  to  inhibit  the 
growth  of  the  organism.  If  the  chemical  substance  is  highly 
poisonous  and  is  present  in  sufficient  amount  it  will  event- 
ually kill  the  bacteria  present.  On  the  other  hand,  many 
weak  substances,  commonly  designated  as  preservatives, 
will  prevent  the  development  of  bacteria,  but  are  not  able 
to  destroy  them.  It  is  necessary,  therefore,  to  clearly  un- 
derstand the  distinction  between  an  antiseptic  and  a  germicide. 
The  latter  kills  a  growth,  whereas  the  former  prevents  its 
further  development.  A  germicide,  when  sufficiently  dilu- 
ted, may  act  as  an  antiseptic.  When  an  agar  tube,  inocu- 
lated with  the  spores  of  the  hay  bacillus,  is  exposed  to  an 
atmosphere  of  sulphur  dioxide  it  becomes  cloudy,  and  if  it 
is  then  placed  in  the  incubator  no  growth  will  result.  Ap- 
parently the  organism  has  been  killed  by  the  sulphur  diox- 
ide. If,  however,  a  platinum  wire  is  passed  over  the  sur- 
face of  the  cloudy  agar  and  is  then  rubbed  over  a  fresh 
agar  tube,  the  latter  will  show  in  a  few  hours  an  abundant 
growth.  Enough  sulphur  dioxide  was  dissolved  in  the  first 
agar  to  prevent  the  growth  of  the  organisms  on  its  surface. 
It  was  not  strong  enough,  however,  to  destroy  them. 


528  BACTERIOLOGY. 

The  student  should  test  tfte  antiseptic  action  of  several  chem- 
icals, according-  to  the  following  directions,  on  one  of  the  bacteria 
employed  in  the  disinfection  experiments. 

Antiseptic  action  of  mercuric  chloride. — A  bacterial  suspension  is 
made  by  adding-  a  few  drops  of  a  fresh  bouillon  culture  of  the  germ 
to  about  200  c.c.  of  sterile  bouillon.  The  1:  500  mercuric  chloride  solu- 
tion is  also  used.  4  larg-e  sterile  tubes  are  numbered  consecutively, 
and  equipped  as  follows: 

No.  1—1  c.c.  of  the  Hg-Cl2  +  9  c.c.  of  the  suspension  =  1: 5,000. 
No.  2—0.5  c.c.  of  the  Hg-Cla  +  9.5  c.c.  of  the  suspension  =  1: 10,000. 
No.  3-0.25  c.c.  of  the  HgGla  +  9.75  c.c.  of  the  suspension  =  1:  20,000. 
No.  4—0.1  c.c.  of  the  Hg-Cl2  -f  9.9  c.c.  of  the  suspension  =  1:50,000. 

The  tubes  are  then  placed  in  the  incubator  and  examined  at  the 
end  of  24  hours.  From  the  cultures  that  show  no  growth,  or  at  most 
a  very  faint  cloudiness,  a  few  drops  should  be  transplanted  to  sterile 
bouillon  tubes.  The  two  sets  should  be  returned  to  the  incubator  and 
examined  on  the  following  day.  Hanging-drop  preparations  of  the 
two  sets  should  be  made.  Involution  forms  may  be  expected.  The  re- 
sults should  be  tabulated. 

Antiseptic  action  of  carbolic  acid. — An  aqueous  1  per  cent,  solution  of 
phenol  is  placed  in  a  stoppered  flask  or  bottle  and  steamed  for  a  few 
minutes.  The  same  bacterial  suspension  is  used  as  above.  5  sterile 
tubes  are  numbered  and  equipped  as  follows: 

No.  1 — 2  c.c.  of  the  phenol  +  8  c.c.  of  the  suspension  =  1: 500. 
No.  2 — 1  c.c.  of  the  phenol  +  9  c.c.  of  the  suspension  =  1: 1,000. 
No.  3— 0.5  c.c.  of  the  phenol  -+-  9?5  c.c.  of  the  suspension  =  1:2,000. 
No.  4—0.25  c.c.  of  the  phenol  +  9.75  c.c.  of  the  suspension  =  1:  4,000. 
No.  5—0.1  c.c.  of  the  phenol  +  9.9  c.c.  of  the  suspension  =  1: 10,000. 

The  tubes  are  then  placed  in  the  incubator  and  examined  as  in 
the  preceding  experiment. 

Antiseptic  action  of  formaldehyde. — 2.5  c.c.  of  the  commercial  40  per 
cent,  solution  of  formaldehyde  are  diluted  to  100  c.c.  with  sterile 
water  ( =  1  per  cent.).  A  series  of  5  dilutions  is  then  made  employing- 
the  same  quantities  as  in  the  above  experiments. 

Antiseptic  action  of  sodium  benzoate. — An  aqueous  1  per  cent,  solu- 
tion of  this  salt  is  sterilized  by  exposure  to  steam.  The  5  dilutions 
are  prepared  and  tested  in  exactly  the  same  manner  as  in  the  case  of 
carbolic  acid. 


ROOM  DISINFECTION.  529 

Repeated  cultivation  of  an  organism,  generation  after 
generation,  in  the  presence  of  an  antiseptic  such  as  carbolic 
acid  will  result  in  a  more  or  less  profound  alteration  of  the 
physiological  properties  of  the  organism.  Thus,  when  the 
anthrax  bacillus  is  grown  at  32°  in  veal  bouillon,  contain- 
ing variable  amounts  of  phenol  (from  1:  500  to  1:  5,000)  and 
transplantations  are  made  every  8-10  days,  the  sporeless  or 
asporogenic  modification  will  be  obtained.  If  a  similar  culti- 
vation is  carried  on  at  42.5°  not  only  is  the  sporeless  variety 
obtained,  but  this  is  also  deprived  of  its  virulence.  In  other 
words,  the  antiseptic  acting  at  a  higher  temperature  in- 
duces more  marked  alterations  than  at  a  lower  temperature 
and  hence  give  rise  to  sporeless,  attenuated  cultures.  The 
asporogenic  characteristic  is  permanent  on  ordinary  media 
but  the  spore  production  is  said  to  return  after  cultivation 
on  peptonless  agar. 

Similar  modifications  are  obtained  by  growing  motile 
bacteria  in  media  containing  carbolic  acid.  Thus,  the 
Eberth  and  colon  bacillus  will  lose  their  motility,  when 
carried  through  several  generations  at  39°,  in  bouillon  con- 
taining about  1  :  3000  of  carbolic  acid. 

Room  Disinfection. 


In  this  laboratory  a  special  room  is  provided  having  a 
capacity  of  1,016  cu.  feet  (28.8  cu.  m.)  in  which  gaseous  dis- 
infectants may  be  tested.  The  gaseous  compounds  com- 
monly in  use  at  the  present  time  are  sulphur  dioxide  and 
formaldehyde. 

The  sulphur,  either  in  the  form  of  flowers  or  in  rolls,  is 
placed  in  an  iron  water-bath  which  is  set  over  a  large  pan 
of  water.  3  pounds  of  sulphur  per  1,000  cu.  feet  are  ordin- 
arily employed.  .  To  this  amount,  50  c.c.  or  more  of  alcohol 
are  added  and  this  is  then  set  on  fire.  The  specimens  have 
previously  been  arranged  in  the  room,  under  the  desired  ex- 
perimental conditions.  All  cracks  about  the  door  and  win- 

34 


530 


BACTERIOLOGY. 


dows  are  securely  caulked  with  strips  of  cloth,  or  better 
with  putty.  The  usual  time  of  exposure  is  20  hours. 

The   formaldehyde   experiments   may   be   carried   out, 
either  by  sprinkling  large  sheets  of  muslin  or  filter-paper 

with  the  necessary  amount  of 
the  40  per  cent,  solution  and 
then  hanging  these  up  in  the 
room,  or  by  distilling  the  for- 
maldehyde into  the  room 
through  a  tube  inserted  into 
the  key-hole.  The  author's 
apparatus1  shown  in  Fig.  76 
has  been  especially  designed 
for  practical  room  disinfec- 
tion. The  copper  vessel  can 
be  heated  with  a  Bunsen  burn- 
er or  with  a  Primus  kerosene 
lamp.  150  c.c.  of  the  40  per 
cent,  formaldehyde  solution 
are  used  for  each  1,000  cu.  ft. 
of  air  space. 

FIG.  76.     The  author's   formaldehyde  ap-  ry,,       ,  .     , 

paratus  for  room   disinfection      a— Narrow  The  bacterial  Suspensions 

tube,  to  be  inserted   in  key-hole;  b — Funnel 

tube  provided  with  solid  stopper;  c— Reser-  are  prepared  according  to  the 

voir;  d — Kerosene  burner. 

directions    given    on    p.    514; 

and  sterile  silk-threads,  muslin  squares  and  cover-glasses 
are  then  infected.  The  action  of  the  disinfectant  should  be 
studied,  at  the  same  time,  on  moist  and  on  dry  specimens. 
The  moist  specimens  can  be  kept  in  this  condition,  for  hours 
if  need  be,  by  placing  them  over  water  in  a  large  moist- 
chamber.  The  dried  specimens  are  obtained  by  placing  the 
freshly  prepared  set  in  an  incubator  at  39°  for  2-3  hours,  the 
cover  of  the  dish  being  slightly  ajar.  When  the  specimens 
have  become  dry  they  should  be  loosened  from  the  bottom 
by  means  of  sterile  forceps. 

teacher's  Sanitary  Bulletin,  No.  3,   Michigan  State  Board  of 
Health;  Medical  News,  May,  1898,  p.  641. 


HARDENING  OF    TISSUE.  531 

The- wet  and  dry  specimens  are  exposed  in  open  Es- 
march  dishes  in  the  room  during-  the  disinfection  process. 
At  the  close  of  the  period  of  disinfection,  5,  10,  or  20  hours, 
the  room  is  entered  and  the  covers  promptly  replaced. 
Each  specimen  is  then  taken  up  by  a  pair  of  forceps  and 
transferred  to  a  tube  of  bouillon.  The  forceps  must  be 
sterilized  in  the  flame  before  making  each  transfer. 

The  student  will  make  experiments  in  the  manner  indi- 
cated with  anthrax  spores,  diphtheria  and  typhoid  fever 
bacilli,  and  with  staphylococci.  The  results  should  be 
carefully  controlled  as  indicated  on  p.  523. 

Hardening,  Imbedding  and  Cutting  of  Sections. 

The  direct  microscopical  examination  of  streak  prepar- 
ations made  from  the  organs  and  tissues  of  infected  ani- 
mals, as  well  as  cultural  experiments  will,  as  a  rule,  reveal 
the  presence  of  micro-organisms.  In  order  to  ascertain  the 
presence  and  especially  the  distribution  of  organisms  within 
the  tissues  and  organs  it  is  necessary  to  harden  these,  then 
.to  cut  sections  and  finally  to  stain  the  sections  by  suitable 
methods. 

The  tissue  to  be  hardened  must  be  cut  up  into  small 
pieces,  about  5-8  mm.  in  thickness.  In  special  cases  even 
thinner  pieces  must  be  used.  These  are  then  placed  in  the 
fixing  and  hardening  fluid.  It  is  always  advisable  to  place 
the  pieces  of  tissue  on  a  piece  of  filter-paper  or  on  some  ab- 
sorbent cotton.  The  liquid  thus  has  free  access  to  all  parts 
of  the  tissue.  The  fixing  and  hardening  of  tissue,  which  is 
to  be  stained  for  bacteria,  is  usually  done  in  alcohol,  mer- 
curic chloride  or  in  a  formaldehyde  solution.  Small  wide- 
mouth  bottles  should  be  employed. 

Alcohol. — The  pieces  of  tissue,  supported  on  filter-paper 
or  cotton,  are  placed  direct  in  95  per  cent,  alcohol.  They 
are  allowed  to  remain  in  this  alcohol  for  3  or  4  days,  after 


532  BACTERIOLOGY. 

which  they  are  transferred  to  absolute  alcohol  for  1  to  2 
days.  In  case  the  pieces  of  tissue  are  large  it  will  be  well 
to  make  a  second  transfer  to  absolute  alcohol. 

The  tissues  maybe  placed  in  alcohol  containing-  2.5  per 
cent,  of  the  commercial  formaldehyde  (1  per  cent,  of  the 
gas).  This  may  be  especially  desirable  when  the  tissue 
contains  highly  virulent  organisms. 

Mercuric  chloride.  — A  saturated  aqueous  solution  of  this 
salt  is  used.  An  addition  of  5  per  cent,  of  glacial  acetic 
acid  may  be  made.  The  tissue  is  fixed  in  this  solution  in 
from  4  to  12  hours.  It  may,  however,  be  left  in  the  liquid 
for  24  hours.  The  tissue  must  then  be  thoroughly  washed 
in  running  water.  This  is  done  by  placing  the  bottle,  the 
mouth  of  which  is  covered  with  a  piece  of  wire  gauze,  under 
a  hydrant  for  12  to  24  hours.  The  pieces  of  tissue  are  then 
placed  in  70  per  cent,  alcohol  for  24  hours,  after  which  they 
are  transferred  for  a  like  period  of  time  to  95  per  cent,  and 
finally  to  absolute  alcohol. 

A  serious  draw-back  to  the  use  of  mercuric  chloride  is 
its  tendency  to  deposit  a  black,  granular  or  semi-crystalline 
precipitate  in  the  tissue.  The  granules  should  not  be  con- 
founded with  micrococci. 

Formaldehyde. — A  4  per  cent,  solution  of  formaldehyde 
can  be  used  for  hardening  tissue.  This  is  prepared  by 
adding  one  part  (10  c.c.)  of  the  commercial  40  per  cent, 
solution  of  formaldehyde  to  9  parts  (90  c.c.)  of  distilled 
water.  The  tissue  is  allowed  to  remain  in  this  solution  for 
4  to  12  hours.  It  is  then  transferred  to  70  per  cent,  alcohol 
for  24  hours,  after  which  it  is  placed  for  a  like  period  of 
time  in  95  per  cent,  and  finally  into  absolute  alcohol. 

It  will  be  seen  that  no  matter  what  solution  is  used  for  fixing, 
eventually  the  tissues  are  placed  in  absolute  alcohol.  When  thor- 
oughly dehydrated  the  material  is  now  ready  for  cutting-  direct,  or 
for  imbedding  and  subsequent  cutting.  If  the  tissue  is  to  be  kept  for 


IMBEDDING  OF  TISSUE.  533 

some  time  it  is  advisable  to  preserve  it  in  a  dilute  alcohol  of  about  70 
per  cent,  strength.  Many  bacteria,  however,  are  affected  by  pro- 
longed sojourn  in  alcohol  to  such  an  extent  that  they  will  not  readily 
stain.  This  is  notably  true  of  the  leprosy  and  tubercle  bacilli.  The 
author  prefers,  therefore,  to  imbed  the  tissue  in  paraffin  and  keep  it 
in  this  form.  The  paraffin  with  the  tissue  may  be  kept  in  bottles 
rather  than  be  put  up  into  blocks. 

Imbedding  in  paraffin. — The  first  step  toward  imbedding" 
in  paraffin  is  to  place  the  tissue  from  absolute  alcohol  into 
toluol,  for  24  hours,  then  for  a  like  period  into  a  strong- 
solution  of  paraffin  in  toluol.  Xylol,  chloroform  or  tur- 
pentine may  be  used  in  place  of  toluol. 

The  paraffin  necessary  for  the  next  step  is  kept  in  a 
melted  condition  in  a  suitable  oven.  An  ordinary  air-bath 
may  be  used,  although  it  is  better  to  employ  one  with  a 
water-jacket.  The  temperature  of  the  oven  should  be 
about  50°,  and  is  controlled  by  a  thermo- regulator  (p. 
246).  Two  wide-mouth  bottles  should  contain  the  necessary 
soft  and  hard  paraffin.  The  soft  paraffin  melts  at  from  38  to 
42°,  whereas  the  hard  paraffin  melts  at  about  46°.  The 
latter  is  usually  prepared  by  bringing  together  equal  parts 
38  and  -52°  paraffin.  In  very  warm  weather  it  will  be  neces* 
sary  to  use  two  parts  of  the  latter  to  one  of  the  former. 

The  tissue,  after  permeation  with  the  toluol  paraffin 
mixture,  is  placed  in  a  small  wide-mouth  bottle,  or  better 
in  the  so-called  tube  vials.  It  is  covered  with  melted  soft 
paraffin  and  placed  in  the  oven  for  12  to  24  hours.  The 
soft  paraffin  is  then  replaced  by  the  melted  hard  paraffin 
which  is  also  allowed  to  act  for  12  to  24  hours.  The 
tissue  now  thoroughly  permeated  with  hard  paraffin  is 
ready  to  be  blocked. 

A  rectangular  trough,  made  by  bringing  together  two 
glass  L's  on  a  zinc  plate,  is  filled  with  melted  hard  paraffin. 
When  the  paraffin  has  slightly  congealed  on  the  bottom  of 
trough,  the  piece  of  tissue  is  introduced  into  the  liquid  by 
means  of  a  previously  warmed  pair  of  forceps.  As  soon  as 
the  paraffin  has  cooled,  so  as  to  become  opaque,  the  plate  is 


534  BACTERIOLOGY. 

placed  in  cold  water.  The  paraffin  now  sets  thoroughly, 
and  after  a  few  minutes  the  L's  can  be  removed.  The 
block  is  now  ready  to  be  cut. 

When  a  number  of  paraffin  blocks  are  prepared,  and  especially 
if  they  are  to  be  kept  for  some  time,  they  should  be  labeled.  This 
can  be  done  by  placing  the  label,  with  the  written  side  turned  down, 
in  the  bottom  of  the  trough  before  pouring-  in  the  paraffin.  The  label 
then  adheres  to  the  paraffin  block.  V  -.  "' 

Imbedding  in  celloidin.- — The  tissue  is  transferred  from 
absolute  alcohol  to  a  mixture  of  equal  parts  of  absolute 
alcohol  and  ether  for  24  hours.  It  is  then  placed  in  ether  for 
12  hours,  and  from  this  it  is  transferred  to  thin  celloidin 
where  it  remains  for  2  to  3  days  or  longer.  The  tissue  is 
then  placed  in  thick  celloidin  for  an  equal  length  of  time 
after  which  it  is  ready  to  be  blocked. 

The  end  of  a  cork  or  similar  block  of  wood  is  covered 
with  thick  celloidin.  This  is  allowed  to  partially  evapor- 
ate and  the  tissue  is  then  placed  on  the  block.  After  par- 
tial drying  in  the  air  the  block  is  placed  in  80  per  cent,  al- 
cohol for  24  hours.  The  material  can  then  be  sectioned  or 
it  may  be  preserved  in  this  alcohol  for  future  use. 

Ordinary  collodium  may  be  used.  The  thick  solution  can  be 
readily  obtained  by  allowing1  some  of  the  collodium  to  remain  in  an 
open  wide-mouth  bottle  for  some  hours.  The  material  can  be  thinned 
whenever  necessary  by  the  addition  of  a  mixture  of  equal  parts  of  ab- 
solute alcohol  and  ether. 

Cutting  sections. — A  good  sliding  microtome  should  be  on 
hand.  This  can  be  used  for  sectioning  the  material  which 
has  been  frozen,  hardened  in  alcohol,  or  imbedded  in  paraf- 
fin or  celloidin. 

The  tissue  which  has  been  fixed  and  hardened  in  alco- 
hol can  be  cut  direct  without  resorting  to  the  imbedding 
process.  The  result,  however,  cannot  be  said  to  be  as 
good.  To  do  this  a  piece  of  the  tissue  is  attached  to  a  small 
cork  by  means  of  a  glycerin  gelatin  mixture  made  by  warm- 


CUTTING  OF  SECTIONS.  535 

ing  1  part  of  gelatin,  2  parts  of  water  and  4  parts  of  gly- 
cerin. The  cork  is  then  securely  clamped  to  the  microtome 
and  the  sections  are  cut.  The  tissue  and  knife  must  be  kept 
moistened  with  alcohol  and  the  sections  are  transferred  at 
once  to  alcohol  by  means  of  a  camel-hair  brush. 

Frozen  sections. — The  tissue  fixed  and  hardened  in  alco- 
hol may  be  cut  by  means  of  the  freezing  microtome.  It 
should  not  be  more  than  2  or  3  mm.  thick.  The  alcohol 
must  first  be  removed  from  the  hardened  tissue  by  immer- 
sion in  water.  This  can  be  accomplished  in  cold  water  in 
from  4  to  8  hours,  depending  on  the  size  of  the  piece.  If  the 
water  is  warmed  to  38°  the  alcohol  will  be  remove'd  more 
rapidly,  in  from  1  to  2  hours.  The  tissue  can  then  be  frozen 
by  the  ether  spray  apparatus  and  sections  cut.  The  knife 
is  moistened  with  water  and  each  section  is  at  once  trans- 
ferred to  water.  This  method  is  very  useful  in  the  staining 
of  bacteria  in  tissues. 

Paraffin  sections. — The  paraffin  block  is  trimmed  square 
and  then  firmly  attached  to  the  metal  holder.  When  cut- 
ting paraffin  sections  the  knife  must  be  kept  perfectly  dry. 
Moreover,  the  edge  of  the  knife  should  be  parallel  to  that 
of  the  block.  In  other  words,  the  knife  is  not  fixed  in  a 
slanting  position  as  in  the  case  of  alcohol  hardened  or  cel- 
loidin  imbedded  objects. 

This  method  of  imbedding  and  cutting  sections  is  easy 
of  execution  and  thinner  sections  can  be  obtained  than  by 
any  other  procedure.  It  is  to  be  preferred  to  the  others 
for  making  bacteriological,  examinations  of  tissue.  The 
paraffin  sections  are  transferred  from  the  knife  by  means  of 
a  brush  or  needle  to  a  piece  of  clean  filter-paper,  which  is 
covered  by  a  bell- jar.  Before  staining  the  sections  it  is,  as 
a  rule,  necessary  to  remove  the  paraffin.  This  can  be 
readily  done  by  means  of  toluol,  xylol  or  turpentine.  The 
sections,  contained  in  an  Esmarch  dish,  should  be  washed 


536  BACTERIOLOGY. 

several  times  with  the  solvent  to  insure  complete  removal 
of  the  paraffin.  The  washed  sections  can  then  be  bottled 
in  70  per  cent,  alcohol. 

The  paramn  should  not  be  removed  from  the  sections  in 
the  manner  described  unless  these  are  sufficiently  thick  to 
hold  together.  Very  thin  sections  are  liable  to  fall  to 
pieces,  and  moreover,  are  difficult  to  handle  in  the  sub- 
sequent process  of  staining-.  In  such  cases,  it  is  advis- 
able to  fix  the  section  to  a  cover-glass  before  removing 
the  paraffin.  This  can  be  easily  done  by  the  following- 
method: 

The  paraffin  sections  frequently  become  curled  or  folded. 
This  difficulty  can  be  readily  overcome  by  placing-  the  sec- 
tions in  some  tepid  water  contained  in  a  larg-e  evaporating 
dish.  The  water  must  not  be  so  warm  as  to  melt  the  paraf- 
fin. The  sections  promptly  spread  out  on  the  surface  of 
the  water.  They  are  now  ready  to  be  taken  up  on  cover- 
glasses.  For  this  purpose  only  perfectly  clean  cover- 
glasses  (p.  140)  should  be  used. 

A  minute  drop  of  the  albumin  fixative  is  placed  on  the 
cover-glass  and  spread  out  in  a  very  thin  layer.  The  cover- 
glass,  thus  prepared,  and  held  in  a  pair  of  forceps,  is  placed 
under  the  floating  section.  In  this  way  the  section  can  be 
raised  and  removed  from  the  water.  The  section  should  be 
pressed  out  flat  on  the  cover-glass  by  gently  applying  the 
tip  of  the  finger.  The  cover-glasses,  thus  equipped  with 
sections,  are  now  set  aside  in  the  incubator  at  about  37°  for 
24  hours  in  order  that  the  sections  may  become  firmly  fixed 
to  the  glass.  The  paraffin  can  now  be  removed  from  the 
cover-glass  by  treatment  with  toluol,  or  other .  solvent,  in 
the  manner  indicated  above. 

The  albumin  fixative  is  prepared  by  cutting-  up  the  white  of  an 
egg-  with  a  pair  of  scissors.  The  liquid  is  then  strained  throug-h  mus- 
lin. An  equal  volume  of  glycerin  is  then  added  to  the  filtrate  and 
thoroughly  mixed.  A  piece  of  camphor  may  be  added  to,prevent  the 
development  of  moulds. 


STAINING  OF  SECTIONS.  537 

Celloidin  sections. — In  cutting'  sections  from  the  celloidin 
block  this,  as  well  as  the  knife,  should  be  kept  moistened 
with  dilute  alcohol.  The  sections  can  be  kept  for  a  long  time 
in  70  per  cent,  alcohol.  On  treatment  with  anilin  dyes  the 
celloidin  becomes  slightly  stained  and  for  that  reason  it  has 
been  suggested  to  dissolve  out  the  celloidin,  before  stain- 
ing, as  in  the  case  of  paraffin.  This,  however,  is  wholly 
unnecessary  in  bacteriological  work. 

Inasmuch  as  oil  of  cloves  dissolves  celloidin,  the  sec- 
tions should  not  be  cleared  in  this  oil  but  in  oil  of  origanum. 

The  Staining  of  Sections. 

It  is  very  difficult  at  times  to  demonstrate  the  presence 
of  organisms  in  sections,  although  they  may  be  easily 
shown  to  be  present  in  ordinary  streak  cover-glass  prepar- 
ations. This  is  frequently  due  to  the  absence  of  any  sharp 
means  of  differentiating  the  organism  from  the  surrounding 
tissue.  The  basic  anilin  dyes  employed,  it  should  be  re- 
membered, are  nuclear  as  well  as  bacterial  stains.  The 
organism,  therefore,  has  the  same  stain  as  the  mass  of 
tissue  in  which  it  lies  imbedded.  In  many  cases,  however, 
a  sharp  differentiation  can  be  obtained  by  double  staining 
either  by  Gram's  method;  or,  as  in  the  case  of  leprosy  and 
tuberculosis,  by  the  application  of  the  usual  process  for 
staining  these  bacilli. 

The  student  should  begin  the  staining  of  sections  made 
from  the  kidney,  liver,  spleen  and  lungs  of  a  guinea-pig 
which  died  of  anthrax.  After  acquiring  the  technique 
necessary  for  the  successful  staining  of  bacteria  by  the 
simple  method  and  by  Gram's  process  the  other  special 
stains  can  then  be  taken  up.  The  concentration  of  the 
dye,  time  of  exppsure  and  the  temperature  of  the  liquid  are 
important  factors  which  must  not  be  lost  sight  of  by  the 
operator.  The  concentration  of  the  acid  or  alcohol  which 


538  BACTERIOLOGY. 

is  employed  in  decoloration  and  the  length  of  time  that 
these  are  allowed  to  act  on  the  stained  section  likewise 
affect  the  result.  In  the  event  of  failure  the  student,  there- 
fore, should  ascertain  by  systematic  trial  which  of  these 
factors  is  the  one  at  fault.  Success  in  staining  sections 
requires  an  intelligent  perseverance  in,  and  a  study  of  the 
method  employed. 

As  a  rule,  a  few  sections  should  be  transferred  to  some 
water  in  an  Esmarch  or  Petri  dish.  Owing  to  the  diffusion 
currents  the  sections  spread  out  perfectly.  The  thin  sec- 
tions can  then  be  transferred  by  means  of  a  mounted  needle, 
at  times  assisted  by  a  spatula,  to  the  filtered,  staining  fluid. 
The  latter  contains  either  fuchsin,  gentian  violet  or  methyl- 
ene  blue. 

•(  The  fuchsin  can  be  used  in  dilute  aqueous  solution 
(p.  147),  such  as  is  employed  in  the  simple  staining  of  cover- 
glasses.  Carbolic  fuchsin  (p.  292)  is  used  for  the  simple  and 
double  staining  of  bacteria. 

Gentian  violet  is  employed  in  dilute  aqueous  solution, 
or  as  anilin-water  gentian  violet  (p.  288).  This  dye  stains 
rapidly  and  deeply  and  it  should  not,  therefore,  be  allowed 
to  act  as  long  as  the  other  dyes. 

Methylene  blue  is  a  slow,  weak  stain  and  should  be 
allowed  more  time  to  act  than  either  of  the  preceding.  It 
may  be  used  in  dilute  aqueous  solution  or  as  Loffler's  alka- 
line methylene  blue  solution  (p.  332).  Kiihne's  carbolic 
methylene  blue,  made  by  adding  1.5  g.  of  methylene  blue 
and  10  g.  of  alcohol  to  100  c.c.  of  5  per  cent,  carbolic  acid 
and  heating  till  complete  solution  takes  place,  is  very 
useful. 

ANTHRAX    BACILLUS. 

Simple  stain.  — The  section  is  transferred  from  water  to 
the  dilute  anilin  dye,  and  is  allowed  to  remain  there  for 
from  5  to  15  minutes.  It  is  then  washed  for  2  or  3  minutes 


SIMPLE  STAINING  OP    SECTIONS.  539* 

in  water  in  order  to  remove  the  excess  of  dye.  After  which 
it  is  placed  in  very  dilute  acetic  acid  (1  c.c.  of  the  glacial 
acid  to  1,000  c.c.  of  water)  for  y2-l  minute.  The  tre'at- 
ment  with  acetic  acid  is  not  always  necessary  and  should  be 
avoided  if  possible.  The  section  is  now  placed  in  strong  alco- 
hol for  */2-l  minute,  and  is  then  transferred  to  clean  water. 

It  is  now  taken  up  on  a  spatula,  placed  on  a  slide  with 
a  drop  of  water,  covered,  and  examined  with  a  No.  7  objec- 
tive. This  examination  is  made  in  order  to  orient  oneself 
as  to  the  condition  of  the  specimen.  The  bacteria  should 
be  deeply  stained  and  should  be  differentiated  as  much  as 
possible  from  the  surrounding-  tissue.  If  they  are  feebly 
stained  it  is  unnecessary  to  proceed  with  the  specimen.  If 
the  tissue  is  still  deeply  stained,  thus  masking-  the  bacteria, 
it  should  be  subjected  ag-ain  to  decoloration  with  acetic- 
water  and  alcohol,  and  re-examined. 

When  the  section  shows  the  proper  degree  of  differen- 
tiation, it  should  be  placed  in  absolute  alcohol  for  a  few 
seconds  in  order  to  thoroughly  dehydrate  it.  Inasmuch  as 
this  treatment  with  alcohol  removes  additional  dye,  it  is 
well  to  stop  the  decoloring-  process,  as  given  above,  while 
the  specimen  is  still  slig-htly  over-stained.  The  treatment 
with  alcohol,  when  dehydrating,  will  remove  this  slight  ex- 
cess of  stain  and  thus  complete  the  differentiation. 

The  section  is  then  placed  in  oil  of  cloves,  cedar  or 
origanum  for  some  minutes,  after  which  it  is  transferred  to 
xylol  and  then  placed  on  a  clean  slide.  The  excess  of  xylol 
is  removed  by  the  application  of  a  piece  of  filter-paper.  A 
drop  of  Canada  balsam  is  now  applied,  and  a  clean  cover- 
glass  is  placed  in  position.  Gentle  pressure,  or  slight 
warming  will  cause  the  balsam  to  spread  out  evenly.  A 
full  history  of  the  specimen  should  be  recorded  on  the 
label,  giving  the  name  of  the  organism  present,  the  animal 
and  organ  used,  the  date  and  method  of  preparation. 

Oil  of  cloves  can  be  used  to  good  advantage  in  the 
clearing  up  of  sections.  It  dissolves  some  of  the  stain  and 


540  BACTERIOLOGY. 

thus  assists  in  the  differentiation.  This  is  especially  true 
when  it  is  used  in  Gram's  method.  All  trace  of  the  oil, 
however,  must  be  removed  from  the  section  by  washing-  in 
xylol.  Some  stains,  like  methylene  blue,  are  readily  dis- 
solved by  the  essential  oils,  and  in  such  instances  the  oil 
can  be  omitted.  The  dehydrated  section  in  that  case  is 
placed  direct  in  xylol. 

In  some  instances  the  treatment  with  xylol  is  dispensed 
with.  Thus,  the  section  may  be  transferred  direct  from  oil 
of  origanum  to  a  glass  slide.  After  wiping-  off  the  excess 
of  oil  around  the  specimen,  a  piece  of  filter-paper  is  firmly 
pressed  down  on  top  of  it.  This  is  done  several  times  until 
all  the  oil  has  been  removed.  The  flattened  section  is  now 
treated  with  balsam  and  covered. 

When  transferring-  the  specimen  on  a  spatula,  care 
should  be  taken  to  keep  the  clean  surface  of  the  instrument 
evenly  moistened  with  the  liquid  in  which  the  section  lies. 
The  spatula  is  slipped  under  the  section  which  is  then 
drawn  up  on  the  blade  by  means  of  a  needle.  To  remove 
the  section  from  the  spatula  to  a  liquid  requires  no 
special  care.  When  the  section,  however,  is  to  be  trans- 
ferred to  a  slide  a  drop  of  the  liquid,  xylol  or  oil,  should  be 
first  placed  on  the  center  of  the  slide.  With  the  end  of  the 
spatula  resting-  in  the  drop  the  section  is  drawn  down  by 
the  needle,  till  a  portion  of  it  rests  on  the  slide.  By  hold- 
ing- this  portion  of  the  section  with  the  needle  the  spatula 
can  now  be  easily  withdrawn. 

Instead  of  using-  the  ordinary  dilute  anilin  dyes,  Lsffler's 
or  Kiihne's  methylene  blue  and  dilute  Ziehl's  solution  may 
be  used.  Thus,  Pfeiffer  stains  the  section  for  half  an  hour 
in  dilute  Ziehl  solution,  then  transfers  it  to  absolute  alcohol 
which  is  very  slig-htly  acidulated  with  acetic  acid.  When 
the  originally  dark-red  section  changes  to  a  peculiar  red- 
dish violet  color,  it  is  cleared  up  at  once  in  xylol  and 
mounted  in  balsam. 

In  staining  sections  it  is  advisable  not  to  overstain  too 


GRAM'S  STAINING  OF  SECTIONS.  541 

much.  An  excessive  exposure  to  dye  requires  a  corre- 
sponding- exposure  to  the  decolorizing-  ag-ents,  and,  as 
a  result,  there  is  lack  of  differentiation.  Hence,  the 
section  should  remain  in  the  dye  for  as  few  minutes  as 
possible. 

Double  staining  by  Gram's  method.  — This  is  an  extremely 
useful  method  but  unfortunately  it  is  not  applicable  to  all 
bacteria.  A  list  of  those  bacteria  which  can  be  stained  by 
Gram's  method  is  given  on  p.  289.  The  method  is  easy  of 
execution  and,  when  properly  carried  out,  it  will  give  clean, 
beautifully  stained  preparations. 

Fresh  solutions  of  anilin-water  g-entian  violet  and  of 
iodine  are  prepared^  according-  to  the  directions  given  on 
p.  288.  The  stain  may  be  slightly  warmed,  but  this  is  not 
necessary.  As  stated  above  it  is  desirable  not  to  overstain 
too  much. 

The  section  is  placed  in  the  stain  for  10  to  15  minutes. 
It  is  then  washed  in  water,  or  in  anilin-water,  to  remove 
the  excess  of  dye.  In  this  way,  the  formation  of  unsig-htly 
deposits  in  the  section,  on  subsequent  contact  with  the  iodine 
solution,  is  avoided.  The  section  is  then  placed  in  the 
solution  of  iodine  (Lug-el's)  for  from  3  to  5  minutes.  It  is 
now  transferred  to  absolute  alcohol,  in  which  it  is  gently 
moved  about  till  most  of  the  stain  is  removed.  The  sec- 
tions should  not  be  completely  decolored,  but  should  still 
show  a  distinct  violet  color. 

It  is  now  placed  in  very  dilute  eosin  for  about  ^  min- 
ute. Over- staining-  with  eosin  will  impart  to  the  tissue  a 
deep  red  color  which  will  thus  make  an  unfavorable  con- 
trast for  the  violet  org-anism.  It  is  preferable,  therefore, 
to  stain  with  eosin  so' that  the  tissue  has  a  lig-ht  pink  color. 
Weigert's  picrocarmin  can  be  used  instead  of  eosin.  It 
should  be  allowed  to  act  for  from  3  to  5  minutes.  The  sec- 
tions may  be  first  stained  with  picrocarmin  and  then  sub- 
jected to  Gram's  staining1. 


542  BACTERIOLOGY. 

The  section  is  transferred  from  eosin  to  absolute  alco- 
hol for  1  or  2  minutes.  When  thoroughly  dehydrated  it  is 
placed  in  oil  of  cloves,  and  should  be  allowed  to  remain  in 
this  oil  till  all  the  violet  color  has  been  taken  out  of  the 
section.  The  sections  may  remain  in  this  oil  over  night 
without  removing-  the  stain  from  the  bacteria.  The  section 
is  then  passed  through  two  dishes  of  xylol,  transferred  to 
a  slide  and  mounted  in  Canada  balsam.  The  deep  violet 
organism  should  stand  out  in  bold  relief  against  a  light 
pink  back-ground. 

The  section  when  placed  in  iodine  tends  to  curl  and 
•easily  breaks.  Very  thin  sections,  therefore,  should  be 
fixed  on  a  cover-glass  previous  to  the  exposure  to  iodine. 

The  following-  summary  of  the  simple  and  of  the  Gram 
method  of  staining-  will  be  useful: 

Simple  Stain.  Gram's  Stain. 

Dil.  anilin  stain  (5  to  15  min.).  Anilin-water  gentian  violet, 

Wash  in  Water  (2  to  3  min.).  (10  to  15  min.). 

Acetic  water  (1-1  min.).  Wash  in  water. 

Strong-  alcohol  (i-1  min.).  Iodine  solution  (3  to  5  min.). 

Water  and  examine.  Decolor  in  absolute  alcohol. 

Absolute  alcohol  (few  sec.).  Very  dilute  eosin  (i  min.). 

Oil  of  cloves,  or  cedar.  Dehydrate  in  abs.  alcohol, 

Xylol.  (1  to  2  min.). 

Mount  in  Canada  balsam.  Oil  of  cloves  (till  decolored). 

Xylol. 

Mount  in  Canada  balsam. 

The  process  of  simple  staining-  is  a  general  method 
which  is  appliable  to  nearly  all  bacteria.  It  must  there- 
fore be  resorted  to  whenever  the  org-anism  does  not  take  the 
Gram's  stain  (p.  290). 

In  some  instances,  where  Gram's  method  fails  owing-  to 
the  removal  of  the  dye  from  the  org-anism  by  the  alcohol, 
the  so-called  WeigerV s  fibrin  stain  can  be  used  to  advantag-e. 
The  cover-glass  preparations  or  sections  are  stained  for  5 
minutes  or  more  in  anilin-water  gentian  violet,  then  rinsed 


TUBERCLE   BACILLUS  IN  SECTIONS.  543 

in  water  and  exposed  to  Lugol's  iodine  solution  (p.  288),  for 
3  to  5  minutes.  The  specimens  are  then  washed  with  water, 
dried  with  filter-paper,  and  transferred  to  a  mixture  of  -2 
parts  of  xylol  and  1  part  of  anilin.  They  remain  in  this 
mixture  till  the  color  ceases  to  be  given  off,  after  which 
they  are  again  dried  with  paper,  covered  with  Canada  bal- 
sam and  examined. 


TUBERCLE    BACILLUS. 

Thin  sections  made  from  tubercular  human  lung-  and 
from  the  spleen,  liver  and  mesenteric  tubercles  of  a  guinea- 
pig  which  received  an  intraperitoneal  injection  of  tubercu- 
lar sputum,  are  stained  according  to  the  following  method, 
which  is  a  modification  of  the  Ziehl-Neelsen  process.  The 
sections,  if  very  thin,  should  be  fixed  on  cover-glasses 
(p.  536). 

It  may  be  well,  first  of  all,  to  call  attention  to  two  con- 
ditions which  influence  the  staining  of  the  tubercle  bacillus. 
In  the  first  place,  the  bacillus  loses  its  specific  staining 
power  when  the  tissue  has  been  kept  in  alcohol  for  some 
time.  This  is  equally  true  of  the  leprosy  bacillus.  It  is 
therefore  advisable  to  use  fresh  tissues;  or,  as  stated  on  p. 
533,  to  preserve  it  in  paraffin.  Again,  Ziehl's  carbolic  fuchsin 
solution  is  not  as  permanent  as  it  is  commonly  said  to  be. 
In  time,  a  tarry  deposit  forms  in  the  bottle  and  the  stain- 
ing power  of  the  liquid  is  materially  decreased.  A  fresh 
Ziehl  solution  should  be  prepared  according  to  the  direc- 
tions given  on  p.  293. 

The  section  is  floated  on  cold,  fresh  carbolic  fuchsin  over 
night;  or,  for  about  -J-  hour  on  a  stain  previously  warmed  to 
about  40°.  The  stain  can  be  heated  on  an  iron-plate  as 
shown  in  Fig.  22.  It  is  then  poured  into  a  warm  Petri  dish. 
The  more  deeply  the  section  is  stained  the  more  difficult  it 
will  be  to  properly  decolor  it. 


544  BACTERIOLOGY. 

The  section  is  then  transferred  to  a  dish  containing 
water  in  order  to  remove  the  excess  of  dye.  It  is  now 
placed  in  60  per  cent,  alcohol  for  1-2  minutes,  and  then  into 
Ebner's  solution  for  -J-  minute,  after  which  it  is  returned  to 
60  per  cent,  alcohol  for  another  minute  or  two,  or  until  it  is 
almost  wholly  decolored.  The  light  pink  color  of  the  sec- 
tion will  be  displaced  on  staining  with  methylene  blue. 

The  almost  decolored  section  is  placed  in  Loffler's 
methylene  blue  for  £  minute,  after  which  it  is  washed  in 
water.  It  is  then  placed  in  absolute  alcohol  for  about  20 
seconds  in  order  to  dehydrate.  A  longer  exposure  to  alcohol 
will  remove  the  blue  stain.  The  section  is  drained  by  plac- 
ing the  edge  of  the  spatula  or  of  the  cover-glass  against 
some  filter-paper;  after  which  it  is  placed  in  xylol  for  2 
minutes.  After  removal  of  the  excess  of  xylol  by  means 
of  filter-paper  the  section  is  mounted  in  Canada  Balsam. 

Cedar  or  anise  oil  may  be  used  to  clear  the  blue  sec- 
tions. On  the  other  hand,  oil  of  cloves  should  be 
avoided  because  it  removes  the  blue  color.  A  properly 
stained  section  will  show  deep  red  bacilli  on  a  light  blue 
back-ground. 

Ebner's  solution  is  ordinarily  used  for  decalcifying  pur- 
poses. The  acid  and  alcohol  present  make  it  very  useful 
for  decolorizing  sections,  and  it  is  to  be  preferred  to  the 
common  procedure  of  treatment  with  nitric  or  sulphuric 
acids.  It  is  prepared  according  to  the  following  formula: 

Sodium  chloride,  0.5 
Hydrochloric  acid,  0.5 
Distilled  water,  30.0 
Alcohol,  100.0 

Instead  of  using  Ebner's  solution  for  decoloring  the 
sections,  a  2  per  cent,  aqueous  solution  of  anilin  hydro- 
chloride  can  be  employed  with  excellent  results  as  it  has 
little  or  no  tendency  to  decolor  the  tubercle  bacilli  (Kiihne, 
Borrel).  The  sections  can  be  first  stained  in  hematoxylin 
or  hematein  for  about  2  minutes.  After  which  they  are 


LEPROSY  BACILLUS   IN  SECTIONS.  545 

washed  in  water  till  they  acquire  the  characteristic  bluish 
tint.  They  are  now  placed  in  Ziehl's  solution  for  about  15 
minutes,  and  then  in  the  anilin  hydrochloride  solution  for  a 
few  seconds.  They  are  now  washed  in  60  per  cent,  alcohol 
till  the  color  ceases  to  be  given  off,  after  which  they  are 
dehydrated  in  absolute  alcohol,  cleared  in  xylol,  and 
mounted  in  balsam. 

The  carbolic  fuchsin  may  be  replaced  by  anilin-water 
fuchsin  or  gentian  violet,  as  employed  in  the  original 
method  of  Ehrlich. 

The  two  methods  for  staining1  tubercle  bacilli  may  be 
summarized  as  follows: 

1st  method.  2nd  method. 

Carbolic  fuchsin  (warm,  15-30  Hematein  (2  min.)- 

min.).  Wash,  and  develop  in  water. 

Wash  in  water.  Carbolic  fuchsin  (15  min.). 

60  per  cent,  alcohol  (1-2  min.).  Anilin  hydrochloride  (few  sec,). 

Ebner's  solution  (%•  min.).  60  per  cent,  alcohol. 

60  per  cent,  alcohol  (1-2  min.)-  Absolute  alcohol. 
Loffler's  methylene  blue  (l/2  min.).    Xylol. 

Wash  in  water.  Canada  balsam. 
Absolute  alcohol  (20  sec.). 
Xylol. 
Canada  balsam. 

LEPROSY   BACILLUS. 


In  sections  made  from  fresh  tissue,  the  leprosy  bacillus 
can  be  stained  by  the  method  employed  for  detecting-  the 
tubercle  bacillus.  But,  as  stated  above,  this  staining-  pe- 
culiarity is  lost,  after  a  time,  if  the  tissue  is  preserved  in 
alcohol.  Although  the  above  method  fails,  yet  the  leprosy 
bacillus  can  be  demonstrated  easily  in  such  tissue  on  stain- 
ing by  Gram's  method  (p.  541).  Under  these  conditions,  ex- 
cellent results  can  be  obtained  by  leaving  the  sections  in 
anilin-water  gentian  violet  over  night. 

The  leprosy  bacillus  is  distinguished  from  the  tubercle 
bacillus  by  being  easily  stained  with  the  ordinary  dilute 


546  BACTERIOLOGY. 

anilin  dyes.  The  simple  stain  method  (p.  538)  can  be  ap- 
plied to  sections  of  the  fresh  tissue  in  order  to  differentiate 
between  the  two  bacilli.  The  enormous  number  of  the 
leprosy  bacilli  and  their  massing-  in  the  so-called  leper 
cells  will  assist  recognition. 

Eberth  Bacillus. 

This  organism,  like  that  of  glanders,  cholera,  etc.,  can- 
not be  demonstrated  in  tissue  by  any  process  of  double 
staining.  In  such  cases  the  simple  stain  method  must  be 
used.  The  sections  should  be  stained  in  LOmer's  alkaline 
methylene  blue  for  some  hours,  then  washed  slightly  in 
water  and  placed  in  a  10  per  cent,  solution  of  tannic  acid. 
The  sections  remain  in  this  mordant  for  from  10  to -60  min- 
utes, after  which  they  are  washed  in  water,  dehydrated  in 
alcohol,  cleared  in  origanum,  and  mounted  in  balsam. 

Actinomyces. 

The  pus  from  an  absc'ess  should  be  received  in  mercuric 
chloride  where  it  soon  hardens.  The  material  after  imbed- 
ding in  paraffin  can  be  cut  into  sections  which  should  be 
fixed  to  cover-glasses.  They  can  be  stained  by  Gram's 
method  or  with  hematoxylin. 

In  the  latter  case,  the  section  should  be  floated  in  a 
Petri  dish  in  water,  to  which  a  drop  or  two  of  Delafield's 
hematoxylin  solution  has  been  added.  It  should  remain  in 
this  stain  for  an  hour  or  more.  Eventually,  it  is  trans- 
ferred to  a  large  volume  of  water  where  it  remains  till  it 
shows  a  clear  blue  color.  The  section  is  then  dehydrated 
in  absolute  alcohol,  cleared  in  oil  of  origanum,  and  mounted 
in  Canada  balsam. 


LIST  OF  APPARATUS  AND  CHEMICALS. 


Anaerobe  tube  apparatus,  p.  314 
Anaerobe  tube  apparatus,  small 
Anaerobe  plate  apparatus,  p.  314 
Anaerobe  plate  apparatus,  vacuum 
Animal  holder,  Latapie's,  p.  268 
Animal  holder,  Voges',  p.  266 
Aspirator,  Chapman's 
Autoclave  20  cm.  diam.,  p.  165 

3  Battery  jars  for  media,  12x12  cm. 

3  Battery  jars  for  media,  18  x  24  cm. 

3  Battery  jars,    15  x  24  cm.   p.  273 
I   Nest  beakers 

1  Blast-lamp,  Fletcher's 

2  Boards,  mounting,  p.  279 

2  Boards,  post-mortem,  p.  276 

8  Bottles  for  stains  Cp.  150),  in  stand 

6  Bottles,  50  c.c. 

6  Bottles,  50  c.c.,  yellow 

4  Boxes  for  slides 

12  Boxes  for  cultures,  p.  512 


2  Burettes,  50  c.c.  in  - 
i  Burette,  100  c.c.,  p. 


c.c.,   p.  155 
511 


100  Filter-paper,  in  circles,  20  cm. 
100  plaited,  32  cm. 

100  hardened,  15  cm. 

2  Filter-plate,  Witte's,  rocm.,  p.  237 

1. 


2  Burette  stands 
2  Burners,  Bunsen 
radial,  p.  263 
safety,  p.  251 
Camera  lucida 
Colony  counter,  p.  434 
paper,  p.  436 
Roll  cotton,  absorbent 

ordinary 
Corks 

200  Cover-  glasses,  No.  i,  15  mm.  diam. 
50  Cover-glasses,  No.  i,  20  mm.  diam. 
i  Cylinder,  graduated,  25  c.c. 
I   Cylinder,  graduated,  50  c.c. 
I  Cylinder,  graduated,    100  c.c. 
I  Cylinder,  graduated,  500  c.c. 
I  Cylinder,  graduated,  1000  c.c. 
I   Disinfecting  jar,  with  top,  15  x  20 

cm.  diam. 
I   Disinfecting    jar,     with    top,    for 

slides,  9  x  10  cm. 
I  Enamelled   jar,  2  1.,  p.  153 

1  Enamelled  stew-pan,  p.  263 

12  Esmarch  dishes,  5  cm.  diam.,  p.  172 
6  6  cm.  diam. 

6  7  cm.  diam. 

2  Files,  triangular 

3  Filter,  Pasteur,  p.  469 

1  Berkefeld,  p.  471 

2  Filtering  cylinder,  Y2  1.,  i  1.,  p.  469 
12  Filter-paper,  in  sheets 


2  Flasks,  Erlenmeyer  vacuum, 
2  Flasks,  Erlenmeyer  vacuum, 
200  c.c.,  p.  469 
200  c.c. 
50  c.c. 

round,  2  1.  capacity 
i       1. 


Roux,  p.  486 
2  Funnels,  15  cm.  diam. 
2  6  cm.  diam. 

2  cylindrical,  p.  514 

I  Forceps,  broad-pointed,  p.  461 
i  cover-glass,  p.  141 

1  narrow  pointed,  p.  461,  locm. 

2  pressure,  p.  461 
i  long,  p.  265 

I  rat,  p.  273 

I  Formaldehyde  generator,  p.  530 
i  Gas  pressure  regulator,  p.  250 
12  Glass  benches,  p.  172 
12  cups  with  covers,  p.  506 

3  globe  receivers,  i  L,  p.  471 
3  containers,  i  1.,  p.  511 

12  plates,  p.  172 

3  rods,  p   172 

loo  slides,  white 

3  concave  well,  p.  143 

500  g.      tubing,  4  mm.  int.  diam.;  wall  i 
"  tubing,  6  mm.          '* 

•'  8  mm.          " 

22  mm.          "  wall  1.5 

3  stop-cocks,  pp.  308,  469 

6  vials  65  X  1  3;  77  X  26  mm. 

Hydrogen  generator,  Kipp's,  i  1. 
Incubator,  high  temperature,  p.  244 
low  temperature,  p.  179 
Instrument  sterilizing  case,  p.  275 
Iron-box  for  pipettes 

for  plates,  5  X  14-  5  X  17-  5  cm. 
plate  for  heating,  p.  150 
water-bath,  18  cm.  diam.  tri- 

pod, p.  150 

100  Labels  for  slides,  white 
12  Liborius  tubes  ' 

4  L's,  glass  or  metal,  p.  533 

I  Micrometer,  cross-wire  ocular 
I  ocular 

i  stage 


548 


LIST  OF  APPARATUS  AND  CHEMICALS. 


I  Microscope,  %,  ^,  TV  objectives; 
2  eye-pieces;  triple  nose-piece, 
Abbe  and  iris  diaphragm 

1  Microtome 

2  Moist  chambers,  p.  172 
I  Nuttall's  needle,  p.  278 

12  Petri  dishes,  p.  172,  180 
6  Pipettes,  i  c.c.  in  y^,  18  cm. 

3  i  c-c-  in  Tta 

3          o.i  c.c.  in  y^,  p.  459 

2,  5,  10,  25  c.c. 


i  Plate,  ice  apparatus  for,  p.  176 

1  water  apparatus,  p.  177 
3  Platinum  wires,  p.  172 

2  Porcelain  evap.  dishes,  1^  1.,  i  1. 

1  Potato  brush 

3  knives 

2  Retort  stands 
loo  Rubber  caps 

2  m.  tubing,  int.  diam.,  6  mm. 

2  m.  tubing,  int.  diam.,  8  mm. 

2  m.  tubing,  vacuum,  int.  diam. 

10  mm.;  wall  4  mm. 
Rubber  stoppers 
2  Scalpels 

2  Scissors,  14  cm. 

curved  on  flat,  12  cm. 
fine  pointed,  10  cm. 
large,  25  cm. 
Searing  iron,  p.  275 
Spatula  for  sections 

Roux,  p.  278 
Speculum,  p.  502 
Sterilizer,  dry  heat,  p.  160 
serum,  Roux,  p.  466 
serum,  Koch,  p.  467 
steam,  p.  164 
Syringes,  i,  2,-  5  c.c.,  p.  262 

holder  for,  p.  263 
Test-glass,  18  cm.  high 
6  30  c.c.  ,  p.  265 

6  50  c.c. 

200  Test-tubes,  12  X  125  mm. 
200  15  X  150  mm. 

20  20  X  150  mm- 

1  brush  or  swab 
6           on  foot,  p.  489 

3  Tubes  for  sacs,  p.  497 

2  Thermometer,  clinical 

1  Kappeler's,  p.  252 

2  280° 

2  IOO° 

2  60°  in  TV 

2  Thermo-regulators,  p.  246 

1  Trephine,  Collin's 

2  Tripods 

6  Tumblers 

1  Wash-bottle,  siphon 

2  Waste  crocks 

6  Watch  glasses,  5  cm. 


1  Wax  pencil,  colored 

2  Wire   baskets,  24  X  32  cm.  diam. 
2  Wire-baskets,  18  X  18  X  24  cm. 

2  Wire-baskets,  10  X  12  X  18  cm. 
2  Wire  cages,  p,  274 

gauze,  1 6  cm.  sq. 

Acetic  acid,  glacial 

Agar  agar 

Alcohol 

Ammonium  hydrate 

Anilin  hydrochloride 

oil 

Bismarck  brown 

Borax 

Canada  balsam  in  tube 

Carbolic  acid 

Celloidin 

Chloroform 

Collodium 

Eosin 

Ether 

Extract  of  meat,  Liebig's 

Ferrous  sulphate 

Fuchsin 

Gelatin 

Gentian  violet 

Glucose 

Glycerin 

Hematein 

Hematoxylin,  Delafield's 

Hydrochloric  acid 

Iodine 

Lactose 

Litmus 


3 

100  g 
100 
1000 
100 

25 

IOO 

25 

200 

20 
IOO 

50 
2OO 
2OO 

25 
200 
IOO 

50 

50 
500 

50 
IOO 
IOO 

25 

IOO 

IOO 

IO 

30 

IOO 

6  sheets 
200 
50 
50 

IOO 

50 
5o 


50 

IOO 

2OOO 

2OO 

IOO 

20 

50 

200 

200 

2OO 

IOO 

50 

500 

500 

IOO 

500 


paper 


Mercuric  chloride 
Methyl  violet 
Methylene  blue 
Nitric  acid 
Oil  of  anise 
cedar 
cloves 
origanum 


'  each  Paraffin,  40°,  49°,  52°,  56° 

liquid 

'       Pepton,  Witte's 
'       Picrocarmin,  Weigert's 
'       Potassium  iodide 
'       Pyrogallic  acid 
'       Sealing  wax 
'       Sodium  carbonate 
'          —  hydrate 
'       Sulphuric  acid 
'       Tannic  acid 
'       Toluol 
'       Turpentine 
'       Vaselin 
Xylol 


i  Kg.  Zinc,  granulated 


INDEX. 


Abbe  condenser,  133,  135 

Abrin,  83 

Abscess,   325,   336,  362,  364,  366,  370, 

374»  384 

Accumulation  method,  430 
Acetic  acid,  90,  96-103 

effect  on  growth,  81 

fermentation,  95,  96,  98 
Acetification,  no 
Acetone,  100 
Achorion,  316,  392,  414 
Achromatic  condenser,  133 

objective,   126 
Acid,  action  of,  77 

dyes,  146 

products,  79,  90,  94,  241 

reaction,  90,  241 

resisting  bacteria,  326-328 
Actinomyces,  317,  416,  546 
Adjustment,  coarse  and  fine,  136 
Aeration,  action  of,  on  toxins,  474 
Aerobic  bacteria,  68 
Aerogenic  bacteria,  92,  93 
Agar-agar,  232 
Agar,  alkalization  of,  238 

colonies  on,  239 

filtration  of,  236 

gelatin,  492,  494 

glycerin,  240 

inclined,  238 

peptonless,  240,284 

plates,  285 

preparation  of,  235,  243 

roll-tubes,  239, 

sedimentation  of,  236 

streak  cultures,  238 
Agglutination,  319,  505 

Testing  of,  506 
Air,  58,  64,  449 

action  of,   76 

analysis  of,  452 

bacteria  in,  451 

city  and  country,  451,  452 

moulds  in,  450 

yeasts  in,  450 


Air,  purification  of,  451 
Albumin,  coagulation  of,  72 

fixative,  536 

Albuminoid  ammonia,  424 
Albumose,  82,  83,  88 
Alcohol,  79,  80,  91,  96-100,  402,  406, 
410 

effect  on  growth,  81 

hardening  with,  531 

effect  on  staining,  320,  533 
Alcoholic    fermentation,    95,    96,    102, 

120,  387 
Aldehyde,  97 
Algae,  117 
Alkali,  89 

Alkaline  reaction,  89-91 
Alkalization  of  media,  154,  234,  238 
Alkaloids,  89 
Amid<o-acids,  91 
Amines,  89-92,  94,  in,  112 
Ammonia,  89,  94,  96,  107-112,  424 
Ammoniacal  fermentation,  107,  424 
Ammonium  chloride,  496 
Amoeba  coli,  372 
Amyl  alcohol,  97 
Amylolytic  ferments,  86,  87 
Anaerobic  apparatus,  306-315 

bacilli,  70 

bacteria,  29,  35,  38,  68,  73,  92,  103, 

163,  298-304 
culture  of,  306 
plate  culture,  312 

micrococci,  70 
Analytic  products,  82 
Angina,  332,  340,  366,  384 
Anilin  dyes,  146 

hydrochloride,  544 

water,  287 

fuchsin,  319 

gentian  violet,   287 
Animal  ferments,  87 

parasites,  259 
Animals,  disposal  of,  383 

cultures  from,  278,  282,  283 

examination  of,  274 


550 


INDEX. 


Animals,  inoculation  of,  260 

observation  of ,  272 
Anthrax,  296 

immunity  to,  489 
Anthrax  bacillus,  76,   194 

poison  of,  83 

sections,  538 

spores,  51,  54,  56,  447 

work  with,  283-294,  538 
Anti-infectious  serum,  484 
Antiseptic,  74,  75,  81,  521,  527 

action  of,  527 

effect  on  motion,  529 

spores,  529 

virulence,  529 

testing  of,  527 
Antitoxin,  484 

action  of,  485,  487 

neutralizing  value  of,  480 

preparation  of,  483 

testing  of,  478,  482 
Aperture,  angle  of,  130 

numerical,  131 
Appendicitis,  352 
Arnold  sterilizer,  163 
Arrows,  poisoned,  298,  300,   304 
Arthrospores,  48 
Artificial  classification,  17 
Ash  of  bacteria,  30,  59,  63 
Ascitic  fluid,  242,  364,  368,  384 
Asparagin,  118,  495 
Aspergillus,  389,  390 

diastatic  action  of,  410 

flavescens,  395,  408 

fumigatus,  395,  410 

infection,  410 

niger,  395,  408 

oryzse,  410 

Asporogenic  bacteria,  51 
Attenuation,   23,   74,   76,  80,   115,  118, 

257'  529 

Autoclave,  75,  165 
use  of,  166 

Bacillus,  17 

aceticus,  99 
acidi  lactici,  226 
aerogenes,  340,  382 
.    •    anthracis,  296 

anthracis  symptomatici,   298 

Asiaticus,  38 

butyricus,  228 

cholerae  gallinarum,  374 

suis,  376 
coli  communis,    38,   62     100,  352 

382 

immobilis,  382 
conjunctividitis,  360 


Bacillus  cyanogenus,  116,  230 
diphtherige,  332 
fluorescens  liquefaciens,  362 

putidus,  204 

icteroides,  38,  356,  382,  492-495 
Indicus,  196 
influenzae,  360 
lactis  aerogenes,  100,  382 
leprse,  320,  326 
leptosporus,  54 
mallei,  330 
megaterium,  54,  216 
mesentericus  vulgatus,  214 
murisepticus,  380 
Neapolitanus,  352 
cedematis  maligni,   300 

No.  II,   302 
ozcense,  340,  342 
Pasteurianus,  99 
pestis  bubonicse,  358,  383 
pneumoniae,  340,  342 
prodigiosus,    160,    170,    17.3,    183, 

194 

psittacosis,   38 
pyocyaneus,  362 
pyogenes  fcetidus,  352 
rabbit  septicemia,  374 
ramosus,  218 

rhinoscleromatis,  340,  342 
rhusiopathiae  suis,  378 
ruber  Kiel,  198 
rubidus,  171,  200 
subtilis,  54,  56,  212 
tetani,  304 

tuberculosis,  322-328 
typhosus,  38,  354 
violaceus,  116,  171,  202      ..  . 
Bacteria,  classification  of,   17,  48 
contents  rf,  30,  34 
defined,  24,  87 
.  distribution,  63 
ends,  19 
examination  of  living,  142,  286 

stained,  149,  286 
form,  17,  18 
function,  93 
liquefying,  86 

multiplication  of,  41,  42,  53,  73,  75 
organisms  smaller  than,  24,  259 
oxygen  requirements  of,  68 
relation  to  disease,  253,  256 
size,  1 8,  24 
staining  of,  31,  145 
structure  of,  25 
thermal  death-point,  513 
.  Bacterial  ferments,  85 
poisons,  83 
proteins,  81,  82 


INDEX. 


551 


Bacterial  suspension,  514,  524 
Bacteriopurpurin,  33,  114 
Bacterium,  17,  18 

aceti,  99 

coli  commune,  352 

phosphorescens,  118,   206 

termo,  114,  22O 

Zopfii,  222 
Bacteroids,  61 
Barley,  88 
Basic  dyes,  146 

products,  84 
Basidia,  391 
Beef  tea,  see  bouillon 
Beer,  96-98,  104,  387 
Beet-juice,  100 
Beggiatoa,  30 
Berkefeld  filter,  464,  471 
Bi-polar  stain,  32,  352,  354,  358,  374 
Biscuit,  slimy,  104 

-shaped  bacteria,  18 
Bismarck  brown,  146 
Black  death,  358,  383 

leg,  298 

plague,  358,   383 
Blastomycetes,  17,  386 
Bleeding  host,  116,  194 
Blight,  392 
Blood,  460,  463 

agar,  360,  364,  368,  384,  463 
plates,  327,  364,  366,  383 

bacteria  in,  65 

coagulation  of,  163,  463 

defibrinated,  489 

drawing  of,  460 

media,  360 

oxalate,  463 

pipettes  for,  458,  460 

plating  of,  327,  383 
Blood-serum,  86,  463,  483, 

boiled,  non-coagulated,  468 

centrifugation  of,  462 

collection  of,  463 

filtration  of,  464 

fractional  sterilization,  464—467 

glycerin,  468 

Loffler's,  468 

preparation  of,  464 

solidification,  467 

see  plasma 

see  serum 

Bloody  milk,  116,  194 
Blue  milk,  230 

pus,  362 
Boiling,  57 

see  moist  heat 
Borax,  275 
Botkin's  method  for  anaerobes,  309 


Botrytis,  391,  392 

Bouillon,  preparation  of.    75,  233,  234. 

243 

blood,  364 

glucose,  243 

glycerin,  240 

Bread-flasks,  104,  116,  394 
Broncho-pneumonia,  336 
Brownian  motion,  34 
Bubonic  plague,  358,  383 
Buchner's  method  for  anaerobes,  307 
Budding  fungi,  17,  305 
Bunt,  392 

Burettes  for  neutralizing,  155 
Burners,  251,  263,  499 
Butter,  flavor  of,  103,  106 

fat,  88,  106 

Butyl  alcohol,  91,  97,  103 
Butyric  acid,  70,  90,  94,  102,  106,  121, 

bacteria,  228 

fermentation,  68,  95,  103 

Cage  for  animals,  274 
Calcium,  51 

acetate,  106 

formate,  106 

hydrate,  105,  284 

lactate,  103 

pectate,  104 
Camera  lucida,  140 
Canada  balsam,  151 
Cancer,  388 

Cane-sugar,  88,  95,  98,  402,  406 
Capillaries  for  thermal  death-point,  459 

emptying  of,  459,  516 

filling  of,   459,  515 

Capsule,   28,  29,    104,   338,   340,    342, 
370 

false,  29,  30 
Carbohydrates,   33,   59,  69,  90-92,  94, 

100,  103,  105,  in,  112 
Carbolic  acid,  51,  56,  81,  91 

antiseptic  action,  528 

disinfecting,  519,  526 

fuchsin,  292,  293,  540 

methylene  blue,  538 
Carbon,  59,   112 

monoxide,  92 
Carbonic  acid,   33,   59,  67,  90,  91,  97- 

101,  103,  105-107,  112,  113,  440 
Caries  of  teeth,  101 

Casein,  102,  104,  106 
Cedar  oil,  130,  137,  145,  544 
Cell  division,  41 

structure,  87 

wall,  25-30 

permeability,  27,  28 
Celloidin,  imbedding  in,  534 


552 


INDEX. 


Celloidin,  sections  537 

Cellulose,  25,  59,  92,  97,  103,  112 

Central  body,  31 

Charbon,  296 

Cheese,  88,  102,  104,  116,  391,  412 

spirillum,   346 
Chemistry  of  bacteria,  79 
Chemotaxis,  78 
Chicken  cholera,  32,  350,  374 

tuberculosis,  328 
Chitin,  26 
Chlorine,  77 

Chlorophyll,  33,  59,  109,  114 
Cholera  Asiatic,  39,  27,  83,  344 

diagnosis,  346 

immunity,  486 

infantum,  see  diarrhea 

infection,  344,  422 

nostras,  348 

poison,  83 

vibrio,  37,  38,  344 
in  discharges,  430 
in  soil,  447 
in  water,  422,  430,  432,  433 

water  relation  to,  422 
Chromatic  aberration,  125 
Chromatin  granules,  28 
Chromogenic  bacteria,  93,  114 
Cider,  98 
Cilia,  38 

Clamps,  313,  470 
Claviceps  purpurea,  392 
Cleavage  products,  82 
Clostridium,  50,  103 
Cloves,  oil  of,  537,  539,  542,  544 
Cocco- bacillus,  17,  358 
Cold,  action  of,  73 
Collodium  sacs.  496 

inoculation  of,  500 

insertion  of,  501 

principle  of,  496 

rolling  of,  497 

sealing  of,  499,  500 
Colon    bacillus,    79,  91,  92,  loi,  102, 
241,  352>  382 

distinctions    from     typhoid,    428, 
492-496 

in  water,  425,  440 
Colony,  169 

deep,  187 

examination  of,  186 

impression,  preparation  of,  222 

isolation  of,  175,  186,  189 

on  agar  plates,  238,  239,  285 
agar-blood  plates;  see  blood 
gelatin  plates,  171,  186 
inclined  agar,  239,  240 
potato,   169,  186 


Colony,  surface,  187 

transplantation,  189 
Columella,  390 
Comma  bacillus,  18,  46,  344 
Compressed  air,  471 
Concave  slide,  143 
Conidia,  389,  391,  393,  402 
Correction  collar,   127 
Cotton  plugs,  158 

spontaneous  combustion,  121 
Counting  of  colonies,  434—437,  448,  449 
Cover- glasses,  140 

cleaning,  141 

for  disinfection,  517,  524 
Cover-glass  forceps,  141 

streaks  from  blood,  280 
cultures,  147 
tissues,  279 
Cream,  106 
Crown-glass,  130 
Cultures,  homogeneous,  514 

sealing  and  keeping,  512 
Cutaneous  inoculation,  261 
Czermak's  holder,  267 

Decalcification,  101 
Decalcifying  liquid,  544 
Deci-normal  alkali,  155 
Deep  layer,  cultures  in,  306 
Defining  power,  129 
Deneke's  vibrio,  346 
De-nitrifying  bacteria,  no 
Desiccation,  55,  58 
Dextran,  105 
Dextrin,  59,  87,  105 
Dextrose,  see  glucose, 
Diaphragm,  iris,  134,  135 
Diarrhea  of  infants,  100,  102,  352 
Diastase,  87,  88,  98 
Diastatic  ferments,  87 

from  moulds,  406-412 
Dilution  cultures,  169 

on  agar,  238,  239 

on  blood-agar,  361 

on  gelatin,  174 

potato,  1 68 
Di-methylamin,  90 
Diphtheria,  52,  83,  332-336 

bacillus,  332,  393 

diagnosis,  334 

immunization,  482 

pseudo-,  335 

streptococci  in,  384 

toxin,  83,  84,  88,  474-476 
fatal  dose  of,  476-478 
test-dose,  481 
Diplo-bacillus,  43 

of  Weeks,  360 


INDEX. 


Diplo-coccus,  44 

gonorrhoeae,  368 

intracellularis  meningitidis,  337 

lanceolatus,  336,  338 

pneumonias,  29,  338,  340 
Disease,  action  of  bacteria  in,  254 

recognition  of  bacteria  in,  255 

relation  of  bacteria  to,  253-259 
Disease-producing  bacteria,  66,  69,  71, 

76,  83,  93 
Disinfectants,  action  of,  527 

errors  in  testing,  518-523 

testing  of,  519,  525 
Disinfection,  factors  involved,  510-523 

of  cover-glasses,  145,  524 

of  hands,  501 

of  rooms,  529 
Division  of  bacteria,  41 
Dough,  rising  of,  98 
Drawing  of  objects,  139 
Draw- tube  of  microscope,  135 
Drum-stick  bacteria,  50,  103,  304 
Dry- heat,  action  of,  56 

sterilization,  1^9,  160 

sterilizer,  159 

testing  of,  517 

Dunham's  solution,  344,  431,  432 
Dust,  73,  452 
Dyspepsia,  101 
Diseases,  bacterial,  258 

fungous,  259,  392 

infectious,  259 

microbic,  259 

protozoal,  259 

with  unknown  cause,  258 

E berth's  bacillus,  see  typhoid  bacillus 

Ebner's  solution,  544 

Egg  albumin,  85 

Electricity,  77 

Eisner's  medium,  490 

Emmerich's  bacillus,  352 

Empusa  muscse,  392 

Empyema,  366 

Endocarditis,  336,  340,  366,  368,  384 

Endospore,  48 

Ensilage,  100,  103,  121 

Environment  of  bacteria,  58 

effect  of,  20 

Enzyme,  59,  85,  116,  396 
Eosin,  146,  289 
Equivalent  focal  distance,  128 
Ergot,  392 
Erysipelas,  364,  384 
Escherich's  bacillus,  352 
Esmarch  counter,  448 

dish,  183 

potato  culture,  183 


Esmarch  counter, 

roll-tube  culture,  181,  239 
Ethyl  alcohol,  91;  see  alcohol 
Eye-piece,  132;  see  ocular 

compensation,  132 

designation  of,  132 

Facultative  bacteria,  67,  69 
Farcy,  330 

in  cattle,  393,  420 

Fat-splitting  ferment,  85,  87,  88,  105 
Fats,  30,  55,  59,  85,  90,  94,  98,  103 

fermentation  of,  105 

rancidity  of,  88,  105,  106 
Fatty  acids,  88,  90,  106 
Favus,  316,  392,  414 
Feces,  29,  427 

Fermentation,   67,    70,    73,   80,  92,  93, 
95,  113,  114,  120,  121 

bacterial,  93 

mould,  391 

putrid,  94,  in 

yeast,  387 

Ferments,  59,  80-82,  85 
Fibrin  stain,  289,  542 
Filamentous  forms.  20 
Filling  of  tubes,  158,  511 
Filter,  absorbent  cotton,  237,  514 

Berkefeld,  464,  471 

•    cleaning  and    sterilizing   of, 
472 

glass-wool,  514 

plaited,  157 

porcelain,  464,  468 

tube,  514 
Filtering  apparatus,  469-471  • 

globe  receiver  for,  471 
Filtration  of  bacterial  liquids,  464,  468 
Finkler-Prior's  vibrio,  348 
Fire-fly,  117 
Fish,  116 

gelatin,  206 
Fission  fungi,  41 
Fixation  of  nitrogen,  no 
Fixing  of  blood  preparations,  280 

cover-glasses,  148 
Flagella.  27,  35 

mordant  for,  318 

see  giant-whips 

stain  for,  319 

staining  of,  316 
Flax,  retting  of,  104 
Flies,  disease  of,  383,  392 
Flint  glass,  126 

Fluorescing  bacteria,  116,  204,  362 
Fluorite  lenses,  131 
Foaming  liver,  92 
Focal  distance,  128 


554 


INDEX. 


Focussing  of  object,  136,  144 
Food  epidemics,  116,  507 

absorption  of,  37 
Forceps  for  cover-glasses,  141 

mice,  265 

pressure,  265,  461 

rats,  273 
Form,  classification    18 

typical,  20 

variation  in,  19,  23 
Formaldehyde,  75,  527 

antiseptic  action,  528 

apparatus,  530 

disinfecting  action,  527 

room  disinfection,  530 

hardening  with,  532 
Formalin,  527 
Formed  ferments,  86 
Formic  acid,  75,  90,  100 
Formless  ferments,  86 
Fox-fire,  117 
Fraenkel's  borer,  448 
Fractional  sterilization,  162,  464 
Fragmentation,  393 
FrankeFs  diplococcus,  338 
Freezing  microtome,  535 
Friedlander's  bacillus,  97,  340,  447 
Fruit-juice,  fermentation  of,  387 
Fruit-organs,  17 

of  moulds,  389—391 
Fuchsin,  146 

anilin- water,  319 

aqueous,  319 

carbolic,  293 

Fungous  diseases,  259,  392 
Fusel  oil,  97 
Fungi,  259,  385 

Gas  pressure  regulator,  250 

Gas  production  by  bacteria,' 70,  79,  91 . 

93,  103,  120,  241 
Gastric  juice,  85,  88 
Geese,  disease  of,  372 
Gelatin,  nutrient,  153,  178 

agar,  492,  494 

alkalization  of,  155 

disadvantage  of,  232 

filtration  of,  157 

inclined,  190 

liquefaction  of,  86,  88,  232 

mineral,  109 

plates,  171 
modified,  179 

potato,  491 

roll-tubes,  181 

stab  cultures,  189 

sterilization  of,  161,  162 

urine,  492 


Generation,  76,  257 
Gentian  violet,  146 

anilin-water,  287 
Germicides,  action  of,  74,  77>  527 

see  disinfectants 

Giant-whips,    38,    208,   298,    304,   309,. 
352,  356 

in  animal  body,  •  39 

in  hanging-drop,  319 
Glanders  bacillus,  52;  59,  330 

diagnosis  of,  329 
Globig's  potato  culture,  184 
Globulin,  83 
Glow-worm  117 

Glucose,  69,  80,  87,  88,  90,  91,  94,  95,. 
98,  100,  101,  103,  105 

agar,  243,  315 

bouillon,  243,  315 

gelatin,  91 

media,  241 

serum,  242 
Glycerin,  88,  91,  96,  98,  105,  106,  118 

media,  240,  243,  315,  472 

mounting  in,  395 
Glycogen,  98,  386 
Glyco-proteids,  25 
Golden  pus  producing  coccus,  366 
Gonococcus,  242,  368 

isolation  of,  384 

media  for,  384 
Gonorrhea,  66,  368 
Gram's  method,  applicability  of,  289 

for  cover-glasses.  287 

for  sections,  541 
Granules,  sporogenic,  31,  32 
Granulose  reaction,  26,  30,  33,  59,  103. 
Grape-juice,  387 
Grape-sugar,  see  glucose 
Green  diarrhea,  204 

pus,  37,  362,  447 

sputum,  204 
Grippe,  360 

Growth,  condition  of,  81 
Gruber's  tubes,  307 
Guinea-pig  cage,  274 

holder  for,  265-268 

inoculation  of,  262,  265 
Halving  process,  95 
Hanging- drop,  143 
culture,  286 
giant-whips  in,  319 
Hardening  of  tissues,  531 
Hay  bacillus,  56,  212 

fermentation  of,  121 

spontaneous  combustion  of,  121 
Heart  blood,  cultures  from,  289 
examination  of,  278,  279 
preservation  of,  279 


INDEX. 


555 


Heart  blood,  streaks  from,  280 
Heat,  action  of,  56,  74,  85 
on  media,  166 

production,  120 
Heating  plate,  150 
Hematein,  544 
Hematoxylin,  544,  546 
Hemp,  retting  of,  104 
Herring  brine,  go 
Herpes,  392,  414 
Hesse's  air  apparatus,  453 
Hiss'  tube  medium,  494 
Hog  cholera,  376 

erysipelas,  378,  447 
Homogeneous  culture,  514 

oil  immersion  lens,  130 
Hops,  fermentation  of,  120 
Horse,  drawing  of  blood  from,  462 

inoculation  of,  263,  268,  269 
Hospital  gangrene,  39 
Hot  springs,  73 
Human  blood  as  medium,  364,  384 

drawing  of,  462 
Hydration  changes,  95 
Hydrochloric  acid,  101 
Hydrogen,   62,   90,    92,    94,    100,  103, 
106,  112 

cultures  in,  308 

generation  of,  312 

peroxide,  76,  77 

sulphide,  m 
Hydrothiohuria,  107 
Hyphse,  389 
Hyphomycetes,  388 
Hypochlorites,  77 

Tee,  442 

Ice  apparatus  for  plates,  175 

Imbedding  in  celloidin,  534 

paraffin,  533 
Immunity,  active,  485 

cause  of,  488 

cholera,  486 

defibrinated  blood,  489 

passive,  485 

unit,  definition,  478 
determination,  479 
neutralizing  value,  480 
Immunization  to  anthrax,  489 

cholera,  486 

diphtheria,  482 

swine-plague,  489 

typhoid  fever,  489 
Impression  preparations,  222 
Incubator,  high  temperature,  71,  243 

low  temperature,    179 

room,  245 
Index  of  refraction,  130 


Indigo,  no 
Indol,  94,  in 

reaction,  79,  344,  428 
Infection,  254,  260 

alimentary,  271 

cutaneous,  261 

ear,  272 

eye,  270 

intra-cranial,  270,  502 

intra-duodenal,  272 

intra-peritoneal,  269,  500 

intra-pleural,  270 

intra-tracheal,  271 

intravenous,  265 

lymphatic,  270 

mixed,  260 

placental,  322 
.  respiratory,  270 

subcutaneous,  262 

subdural,  502 

Infectious  disease,  254,  259 
,Influenza,  360 
Infusoria,  112 

Injection  apparatus,  262,  264 
Inoculation  of  tubes,  173,  174 

with  pipettes,  458 
Insolation,  effect  of,  75,  115 
Insoluble  ferments,  86 
Instruments,  sterilization  of,  167,  275 

sterilizer  for,  275 
Internal  pressure,  26 
Intestinal  bacteria,  65,  66,  69,  88,  92, 

97,  100,  105,  111,352 
Intoxication,  122,  254 
Intracellular  changes,  81 
Invert  sugar,  98 
Invertin,  98,  396 
Inverting  ferment,  87,  88 
Involution  forms,  21,  99,  358,  383 
Iodine,  28,  33,  386 

solution,  288 
Iris  diaphragm,  134 
Isolation  of  bacteria,  169,  171,  189,232, 
253 

Jequirity  seed,  83 

Kappeler's  thermometer,  179 
Kephir,  102 
Kitasato's  flasks,  309 
Klatsch  preparations,  222 
Koch's  dry- heat  sterilizer,  159 

plating  apparatus,  176 

safety   burner,  251 

serum  sterilizer,  466 

steam  sterilizer,  163 
Koumiss,  102 
Kiihne's  methylene  blue,  538 


556 


INDEX. 


Lactic  acid,  81,  90,  94,  100-106,  121 

bacteria,  226 

dextro-rotatory,  101 

fermentation,  95,  100 

inactive,  101 

levo-rotatory,  101 
Lactose,  80,  91,  101,  396 

media,  241 
Lafar's  counter,  436 
Lake  water,  441 
Lanceolate  bacteria,  18,  29 
Latapie's  holder,  267,  268 
Leguminous  plants,  61,  no 
Leprosy,  66,  320,  326 

sections  of,  545 
Leucin,  91 

Leuconostoc,  29,  105 
Leuco-products,  115 
Levulose,  95,  98 
Liborius  tubes,  306,  308,  526 
Liehig's  meat  extract,  243,  474 
Life  history  of  bacteria,  41 
Light,  effect  of  on  bacteria,  75,  85 
on  media,  75 

producing  bacteria,  93 
Lipochrome,  116 
Liquefaction,  88 
Liquors,  96 
Litmus  as  indicator,  154 

media,  91,  96,  240,  241,  310 

purification  of,  509 

sensitizing  of,  510 
Liver,  foaming,  92 
Living  bacteria,  examination  of,  142 

ferments,  86 
Lock-jaw,  304 
Loffler's  bacillus,  332 

methylene  blue,  332 

mordant,  318 

serum,  334,  468 

Low  temperature  apparatus,  179 
Lugol's  solution,  288 
Lumpy- jaw,  316,  393,  416,  546 
Lupus,  322 
Lysol  as  disinfectant,  526 

Madura  foot,  316,  393,  418 
Magnification,  128 

linear,  129 

measurement  of,  128 

superficial,  129 
Malaria,  259,  372 

Malignant  oedema,  91,  194,  300,  446 
No.  II,  302 

pustule,  296 
Mallein,  329,  330 
Malt,  87,  98 
Maltose,  98,  402,  406 


Man,  blood  from,  462 
Mannite,  105 
Marsh-gas,  92,  103 
Martin's  filter,  469 

pepton  solution,  475 
Mass  culture,  168 
Measurement  of  objects,  137 
Meat,  116,  117,  153 
Meat  extract,  Liebig's,  233,  243,  474 

fermented,  474 
Mechanical  interference,  281 
Media,  alteration  by  heat,  166 
light,  95 

sterilization  of,  161,  464 

tubing  of,  511 
Meningitis,  336,  337 
Mercaptans,  94,  in 
Mercuric  chloride,  antiseptic  action  of, 
528 

germicidal  action  of,  525 

hardening  with,  532 

and  hydrogen  sulphide,  525,  526 

solution,  51,  167 

Mesentery,  preparation  from,  281 
Metabolic  products,  79 
Methods  of  infection,  260 
Methylamin,  90 
Methyl  violet,  146 
Methylene  blue,  146 

Kiihne's,  538 

Loffler's,  '332 
Mikulicz,  cells  of,  342 
Mice,  cage  for,  273 

inoculation  of,  262,  265 
Microbic  association,  70,  73,  IO2,  310 

diseases,  259 
Micro- burners,  251 
Micrococci,  forms  of,  46 

motility  of,  35 

multiplication  of,  44 
Micrococcus,  17 

aceti,  99 

gonorrhoese,  368 

pneumonise  crouposse,  338 

prodigiosus,  194 

tetragenus,  370,  447 

urese,  107 
Micrometer,  ocular,  138 

cross- wire,  437 

stage,  137 

screw,  136 

value,  138 

Micro-millimeter,  see  micron 
Micron,  24,  138 
Microscope,  123 

care  of,  136 

Microsporon  furfur,  392,  414 
Mildew,  392 


INDEX. 


55T 


Milk,  65,  88,  103,  116,  230 

alteration  by  heat,  166 

coagulation  of,  102,  429 

examination  of,  455 

fermented,  102 

media,  233,  239 

souring  of,  100,  102,  104,  226 

tubercle  bacillus  in,  325 
Milk  sugar,  see  lactose 
Milzbrand,  296 
Minimum  fatal  dose,  476-478 
Mixed  culture,  152,  310 

infection,  261,  340 
Modified  media,  240 
Moist  chamber,  167,  176 

heat,  56 

testing  of,  515 
Moisture,  58 

Molasses,  fermentation  of,  105 
Molecular  motion,  34 
Monas  prodigiosa,  194 
Monilia  Candida,  388,  392,  402 
Mordant  solution,  318,  546 
Morve,   330 
Mother  of  vinegar,  99 
Motion,  33,  50 

Moulds,    58,  63,   87,  93,  95,  98,  108, 
112,  117,  121,  388 

culture  of,  394 

diseases  due  to,  392 

examination  of,  395 

in  air,  452 

multiplication  of,  16,  389 

structure  of,  389 
Mounting  boards,  279 
Mouse  septicemia,  380 
Mouth  bacteria,  33,  46,  66,  70,  97, 100, 

104,  109,  336,  364,  370 
Mucor;  389,  390 

corymbifer,  393,  404 

fermentation  by,  406 

mucedo,  406 

mycoses,  390,  404 

racemosus,  406 

rhizopodiformis,  395,  406 
Muguet,  402 
Muscardine,  391 
Muslin  squares,  516,  524 
Mycelium,  389 
Mycetoma,  418 
Mycoderma  aceti,  99 

Naphthalene  monobromide,  141 
Natural  classification,  17,  48 
Neisser's  double  stain,  336 
Neutralization  of  media,  154 
Nitric  acid,  96,  108,  112,  424 
v  Nitrification,  108 


Nitrifying  bacteria,  33,  60,  67,  445 
Nitrite  agar,  109 
Nitro-bacteria,  no 
Nitrogen,  60,  92,  108,  112,  113      i 

fixation  of,  no,  445 
Nitroso-bacteria,  no 
Nitrous  acid,  96,  108,  1 1 2,  424 
Non-living  ferments,  86 
Non-pathogenic  bacteria,  71,  72,  193, 
Normal  alkali,  154 
Nose-piece,  136 
Nuclear  matter,  31 

stains,  31 
Nuclein  bases,  31 
Nucleus  in  bacteria,  25,  31 
Nuttall's  needle,  278 

pipette,  modified,  460 

Objectives,  123 

achromatic,   126 

apochromatic,  132,  133 

care  of,  137,  145 

designation  of,  132 

focussing  of,  136,  144 

micrometer  value  of,    138 

oil-immersion,  130 
Obligative  bacteria,  66,  68 
Observation  of  animals,  272 
'Ocean  bacteria,  76,  443 
Ocular,  132;  see  eye-piece 

micrometer,  138 

screw  micrometer,  139 
Oedema,  malignant,  300 

No.  II,  malignant,  302 
Oidia,  414 
Oidium,  389,  391,  392 

albicans,  402 

lactis,  391,  394,  400 

Tuckeri,  400 
Optimum  temperature,  71 
Orange  sarcine,  208 
Organized  ferments,   86,  93 
Origanum,  oil  of,  537 
Osmotic  pressure,  26 
Osteomyelitis,  366 
Otitis  media,  336,  340,  366,  384 
Oxalate  blood  or  plasma,  463 
Oxalic  acid,  go 
Oxidation  changes,  95 
Oxygen,  1 14 

effect  on  growth,  65,  76,  115 
light  production,  118 
sporulation,  51 
toxins,  85 
Oysters,  116 
Ozone,  71 

Palmitic  acid,  90   » 


558 


INDEX. 


Pancreas,  85 

Pancreatic  secretion,  85,  87,   88,  105 

Papain,  87 

Paraffin  bath,  533 

imbedding  in,  533 
sections,  535 
Para-lactic  acid,  101 
Parasitic  bacteria.  66,  71 
Pasteur  filter,  464,  468 

pipette,  456 
Pathogenic  bacteria,  71,  74,  76,  89,93, 

95,  106,  121,  295 
Penetrating  power,  129,  131 
Penicillium,  389,  391 

glaucum,  395  ,412 
Pepsin,  82,  88 
Pepton,  51,  82,  83,  88 

solution,  Martin's,  475 
Witte's,  153 
Peptonizing  bacteria,  101 

ferments,  87 

Peptonless  agar,  240,  529 
Pericarditis,  336,  340,  384 
Perithecia,  390 
Peritoneal  fluid,  277 
Peritonitis,  336,  340,  366,368,  370,384 
Permanent  mounts,  150 
Peronospora  infestans,  392 
Perpetuating  form,  53 
Pest,  358,  446 
Petri,  air  apparatus  of,  454 
dish,  1 80 

culture,  1 80 

Pfeiffer's  reaction,  487,  488 
Phagocytes,   293 
Phenol,  see  carbolic  acid 
Phenol-phthalein,  154 
Phosphorus,  92 
Phosphorescence,  116,  206 
Phosphorescing  animals,  117 
bacteria,  72,  93,  117,  206 
moulds,  117 

Photobacterium,  118,  206 
Photogenic  bacteria,  93 
Physical  motion,  34 
Pickles,  100 
Picro-carmin,  289,  541 
Pigment  bacteria,  76,  93 
primary,  224 
production,  9,  23,  114 
secondary,  115 
Pipette  bottle,  147,  150 

for  drawing  blood,  460 
Pasteur,  456 

advantages  of,  458 
sealing  of  cultures  in,  458 
for  thermal  death-point,  459,  516 
use  of,  277-279 


Pipette, 

for  water  analysis,  433,  459 
graduated,  459 
sterilization  of,  460 
Pityriasis,  392,  414 
Plague,  358 
Plant  diseases,  259 

ferments,  87 
Plasma,  oxalate,  463 
Plasmodium,  372 
Plasmolysis,  22,  27,  32,  36,  352 
Plates,  culture  on,  171 

sterilization  of,  172 
Platinum  wires,  172 
Pleomorphism,  22,  432 
Pleuritis,  336,  340,  384 
Pleuro-pneumonia,  24 
Pneumo-bacillus,   340 
Pneumo-enteritis,  376 
Pneumo-coccus,  no,  338,  340 
Pneumonia,  97,  337,  338,  340,  384 
diagnosis,  336,  337 
in  pest,  383 

secondary  infection  in,  384 
Poison-producing  bacteria,  93 
Poisoned  arrows,  298,  300,  304 
Poisonous  food,  507 

bacterial  products,  83,  84,  88,  106 
Poisons,  relation  to  disease,  254 
Polar  bodies,  27,  32,  336 
Pollution  of  water,  423—425 
Post-mortem  examination,  275 
Potato  bacillus,  214 

spores  of,  162 
culture  on,  167 
dishes,  Esmarch's,  182 
fermentation  of,  120 
gelatin,  490 
glycerin,   240 
tubes,  183 

Precautions  in  laboratory,  170,  282 
Press,  490 

Pressure,  action  of,  76 
Primary  products,  82 
Propionic  acid,  90 
Propyl  alcohol,  97 
Protamin,  31 
Proteins,  26,  30,  37,   59,  81-83,  88,  90, 

92,  94,  103,  106-108,  in,  112 
Protein  free  media,  495 
Proteolytic  ferments,  85-88 
Proteus  vulgaris,  38,  220 
Protoplasm,  25,  30-33,  58,  61,   73,   8l, 

112,     IIQ 

Protozoa,   117 
Protozoal  diseases,  259 
Pseudo-diphtheria,   384 
bacillus,  335 


INDEX. 


559 


Pseud  o, 

flagella,  37 

influenza  bacillus,  360 

spores,  52 

tuberculosis,  326,  328,  393,  420 

typhoid  bacilli,  427 
Ptomains,  84,  88,  90,  122 
Puccinia,  392 
Puerperal  fever,  364,  384 
Punk  fire,  117 

Pure  culture,  isolation  of,  169,  253,  257 
Putrefaction,  67,  93-95,  III,  113,  121 
Putrid  fermentation,  94,  m 
Pyemia,  364,  366,  384 
Pyocyanin,  116,  362 
Pyrogallate  method,  307,  312,  313 

Quarter  evil,  298 

Rabbit  cage,  274 

holder,  267,  268 

inoculation  of,  262,  265,  266 

septicemia,  374 
Rabies,  diagnosis  of,  502 

preservation  of  virulence,  504 

vaccine,  503 

Rag  picker's  disease,  296,  300 
Rain,  65,  441 
Ranvier  slide,  143 
Rat  cage,  273 

forceps  for,  273 

inoculation  of,  262,  265 
Rauschbrand,  298 
Ray  fungus,  416 
Reaction  of  media,  63,  91,  157 

effect  on  color,  115 

phosphorescence,  118 
Real  image,   123 
Recurrent  fever,  46,  372 
Red  bacillus  of  water,  200 

Kiel,  198 
Red  color  on  foods,  194,  200,  208 

milk,  208 

sweat,  208 

yeast,  388,  398 
Reduction  changes,  96 
Relapsing  fever,  372 
Rennet  action,  87,  88,  102 
Reproduction,  47 
Reproductive  form,  47,  53 
Resistance  of  bacteria,  55,  161 

difference  in,  56 

of  spores,  55 

unequal,  in  same  species,  519 
Resolving  power,  129,  131 
Resting  form,  see  spore 
Rhinoscleroma,  342 
Ricin,  83 


River-water,  77,  442 
Rod -shaped  bacteria,  17 
Roll-culture,  agar,  239 

gelatin,  181,  453,  455 

counting  colonies  in,  449 
Rooms,  disinfection  of,  529 

sweeping  of,  452 
Root  bacillus,  218 
Rouget,  378,  380 
Roux  flask,  485 

pipette,  307 

spatula,  278 

syringe,  262 

thermo-regulator,  465 

tube  for  potato,  185,  315 

water-bath,  465 
Rules  of  Koch,  255,  258 

Saccharomyces,  97,  386 

albicans,  402 

cerevisise,  387,  396 

ellipsoideus,  387 

glutinis,  388,  398 

niger,  398 

Pastorianus,  387 
Safety  burner,  251 
Sake,  410 
Saliva,    bacteria  in,    85,   87,  109,  33^- 

338»  366,  370 
Salt,  27,  118,  358 
Salt-peter,  27,  108,  445 
Salts,  inorganic,  62 
Sanarelli's  bacillus,  356 
Sang  de  rate,  296 
Saprogenic  bacteria,  93,  95,  113 
Saprophytic  bacteria,  66,  71,  74 
Sarcine,  44,  45 

motile,  35,  38,  208 

orange,  171,  182,  208 

ventriculi,  210 

yellow,  182,  2IO 
Sarco-lactic  acid,  101 
Sarcoma,  388 
Sauer-kraut,  loo,  103,  121 
Sausage,  104 
Scarlet  fever,  384 
Schizomyce.tes,  41 
Sclerotia,  391 
Screw- shaped  bacteria,  17 
Sealing  of  tubes,  316,  512 
Searing  iron,  275 
Secondary  infection,  256,  384 

products,  82,  96 

spectrum,  126 
Sections,  531 

cutting  of,   534 

fixing  of,  536 

Gram's  stain,  541 


560 


INDEX. 


Sections, 

simple  stain,  538,  540 

tubercle  stain,  543 

Sedgwick-Tucker's  air  apparatus,  454 
Seed,  20,  47,  87 
Segmentation,  393 
Septicemia,  368,  374,  382,  3^3 
Septicemie,  68,  300 
Serum  media.  242 

agar  plates,  242 

agglutination,  319,  487,  505 

anti-infectious,  344,  484,  487 

antitoxic,  344,  478,  483,  484 

Loffier's,  242,  468 

normal.  488,  505 

Pfeiffer's  reaction,  487 

see  blood-serum 

sterilizer,  Koch's,  466 

Roux,  465 
Sewage,  444 
Sheath,  30 
Silk  threads,  517,  523 

worm  disease,  391 
Skatol,  94,  in 
Slimy  beer,  milk,  etc.,  29 

fermentations,  104 
Smegma  bacillus,  326 
Smut,  corn,  392 
Snake  venom,  83 
Snow,  65,  441 
Sodium  benzoate,  antiseptic  action,  528 

carbonate,  154,  275 

hydrate,  154 

deci- normal,  154 
normal,  154 
Soil,  444 

analysis,  448 

bacteria  in,  65,  69,  70,  73,  108 

filtering  action  of,  443 

number  of  bacteria,  446 

pathogenic  bacteria,  446 

vitality  of  bacteria  in,  447 
Soluble  ferments,  86 
Soorpilz,  402 
Species,  identification  of,  19 

origin  of,  23 

variability  of,  23 
Spherical  aberration,  124 

bacteria,  17 
Spirillum,  17,  46 

anserini,  372 

Obermeieri,  372 

rubrum,  115,  224 

see  vibrio 

undula,  27 
Spirochsete,  18,  372 
Splenic  fever,  296 
Spontaneous  combustion,  121 


Spontaneous, 

generation,  56,  161 
Sporangium,  389,  390 
Spores,  characteristics  of,  51 
double  stain,  290 
feebly  resistant,  393 
formation  of,  47—51,  8 1 
germination  of,  52,  75 
of  moulds,  389 
of  streptotrices,  392 
of  yeasts,  386 

resistance  of,  55,  162,  165,  214,  519 
staining  of,  55 
structure  of,  54 

Sporogenic  granules,  31,  32,  48 
Sporulation,  48 
Spring-water,  443 
Sputum  in  influenza,  360 
in  leprosy,  320 
in  pest,  358 
in  pneumonia,  337 
in  tuberculosis,  324,  325 
septicemia,  338 
Stab  culture,  189 
Stage  micrometer,  137 
Staining  cover-glasses,  Gram's,  287 
simple,  145-151,  191,  287 
tubercle  bacillus,  324,  543 
flagella,  316 
glass-slides,  280 
sections,  Gram's,  541 
simple,  538,  540 
tubercle  bacillus,  543 
Stains,  146 

bottle  for,  147,  150 
heating  of,  150 
preparation  of,  146 
Stand  of  microscope,  135 
Staphylococcus,  44,  45 
pyogenes  albus,  366 
aureus,  25,  114,  366 
citreus,  366 
Starch,  86,  87,  98,  112,  115 

splitting  ferments,  85 
Steam-heat,  56,  161 

testing  action  of,  516 
under  pressure,  56,  165 
Steam  sterilizer,   163 
under  pressure,  165 
superheated,  167 
Stearic  acid,  90 
Sterigmas,  390 
Sterile  media,   155 
Sterilization,  159 

at  58°,  fractional,  464 
at  100°,  fractional,  162,  465 
by  filtration,  464 
failure  of,  162 


INDEX. 


561 


Sterilizer,  dry-heat,  159 

see  steam 

Stoddart's  medium,  492 
Stomach,  85,  loo 

bacteria,  66,  92,  101,  102 
sarcine,  210 

Straus-Wurtz  apparatus,  454 
Streak  culture,  186 
on  agar,  238 
agar  blood,  360 
gelatin,  190 
potato,  1 86 

preparations,  fixing  of,  148,  281 
of  blood,  280 

cultures,  147,  148 
organs,  279 

staining  of,  148,  281,  282,  290 
Streptococcus,  44,  45 
erysipelatis,  364 
in  diphtheria,  334 
pyogenes,  364,  383 
Streptothrix,  392 

actinomyces,  416 
farcinica,  420 
Madurae,  418 
Study,  line  of,  191,  192 
Sub-culture,  521 
Succinic  acid,  90,  97 
Sugar,  87,  97,  100,  103-105,  112 

see  glucose,  etc. 

Sulphur  compounds,  90,  92;  107,  in 
dioxide  as  antiseptic,   527 
room  disinfection,  529 
Summer  complaint,  see  diarrhea 
Sunlight,  action  of,  58,  75,  115 
Suppuration,   82 
Surface  tension,  26 
Suspensions,  bacterial,  514,  524 
Swabs,  preparation  of,  334 
Swine  plague,  376 

immunity  to,  489 
Symptomatic  anthrax,  38,  92,  298 
Synthetic  changes,  81,  84 
Syringe,  262-264 
holder,   263 
sterilization  of,  263 
Temperature,  71 

constant,  178,  245 
effect  on  form,  21 
gelatin,   161 
germination,  54 
motion,  35 

multiplication,  41,  65,  71 
phosphorescence,  118 
pigment,  75,  115 
size,  21,  75 
sporulation,  51 
for  cultivation,  74 


Temperature, 

maximum  and  minimum,  71 

of  animals,  273 

optimum,  71 
Test-glass,  265 
Test-tubes,  158,  184,  238 

cleaning  of,  158 

rilling  of,  158,  458 

plugging  of,  158 

sterilization  of,  160 
Tetanus  bacillus,  70,  91,  102,  304,  446 

447 

Tetanus  toxin,  84 
Tetrads,  29,  44,  45 
Thermal  death-point,  513 
Thermogenic  bacteria,  120 
Thermometer,   252 

maximum  and  minimum,  179,  252 
Thermophilic  bacteria,  72 
Thermo- regulator,  245 

alcohol,  246 

filling  of,  248 

mercury,  245 

metallic,  265 
Thermostat,  243 
Thoma  Zeiss  apparatus,  437,  520 
Threads,  20,  43 

for  disinfection,  517,  523 
Thrush,  392,  402 
Tilletia,  392 
Tissue,  normal,  65 

cutting  of,  534 

hardening  of ,  531 

imbedding  of,  533 
Tobacco,  fermentation  of,   120 
Torula,  97,  386,  387 
Toxalbumin,  83 
Toxalbumose,  83 
Toxic,  see  poisonous 
Toxicogenic  bacteria,  93,  121 
Toxin,  59,  81,  84,  88 

diphtheria,  474 

immunizing  with,  482 
Toxoids,  481 
Toxo-pepton,  83 
Trichina,  508 

Tricophyton  tonsurans,  392,  414 
Tri-methylamin,  go,  120,   194 
Trochar,  268,  269,  462 
Trypsin,  88 

Tubercle  bacillus,  30,  32,  52,  59,  65, 
66,  76,  86,  129,241,256,  315, 
322-328,  393 

action  of,  325 

agar  culture,  315 

bouillon  culture,  473 

detection,  324 

differentiation,  326,  327 


562 


INDEX. 


Tubercle  bacillus, 

in  dust,  452 

in  milk,  325 

in  sections,  543 

in  soil,  447 

in  sputum,  324 

in  urine,  325 

potato  culture,  315,  473 

pseudo,  326-328 

see  tuberculosis 
Tuberculin,  322,  326,  472 
Tuberculosis,  aviary,  316,  328 

cattle,  324 

fish,  328 

mammalian,  328 

pseudo,  328 

secondary  infection  in,  384 
Tubing  of  media,  158,  511 
Turnips,  fermentation  of,  120 
Typhoid  bacillus,  354,  382 

behavior  in  water,  426 

detection  in  water,  426,  428 

distinction  from  colon  bacillus,  428, 
489-496 

in  sections,  546 

in  soil  and  feces,  427,  446,  447 

in  water,  427,  428 

toxin  of,  489 

Typhoid  fever,  27,  32,  38,  52,  62,  73, 
79,  86,  91,  92,  97,  101,  102, 
241.  354.  382,  422 

immunity  to,  489 

infection,  382 

secondary  infection,  384 

serum,  action  of,  488,  505-507 
Typical  form,  20 
Ty rosin,  91 

Under-correction,  127 
Unorganized  ferments,  86 
Urea,  89,  95,  107 
Urine,  75,  76,  104,  107,  108 

ammoniacal  fermentation,  107 

bacteria  in,  65,  89,  354 

hydrogen  sulphide  fermentation,  106 

smegma  bacillus  in,   327 

tubercle  bacillus  in,  325 
Uschinsky's  medium,  495 
Ustilago,  392 

Vacuoles,  32,  386 
Vacuum  cultures,  307,  313 
Variability  in  form,  20 

in  species,  23 
Vaughan's  cage,  274 
Vegetating  form,  46,  72-75,   161 
Venom,  83 
Vesuvin,  146 


Vibrio,    18,  46 

cholerse  Asiaticee,  69,  117,  344 

Deneke,  346 

Finkler-Prior,  348 

Metschnikovi,  350 

Miller,  348 

Nordhafen,  350 

phosphorescing,  117 

proteus,  348 
Vibrion  butyrique,  68,  228 

septique,  300 
Vine,  disease  of,  400 
Vinegar,  98 
Violet  bacillus,  202 
Virtual  image,  123 
Virulence,  increase  of,  278,  485,  496 

preservation  of,  279,  332,  364,  458 
Viscose,  105 

Viscous  fermentation,  104 
Vitality  of  cultures,  279 
Voges  holder,  265,  266 

Waste  matter,   112 

Waste  products,  41,  50,  79-81,  89 
of  different  species,  79 
of  weakened  species,  80 

Water,  30,  58,  62,  65,  73,  108 
aerogenic  bacteria  in,  440 
animal  inoculation  with,  440 
cholera  vibrio  in,  429-433 
colon  bacillus  in,  425,  440 
number  of  bacteria  in,  433,  437 
number  of  species  in,  439 
organic  matter  in,  438 
pathogenic  bacteria  in,  439 
relation  to  disease,  422 
typhoid  bacillus  in,  428 
vibrios,  430 

Water  analysis,  422,  433 

bacteriological,  424,  433 
chemical,  422-424 
methods  of,  433 

Water- bath,  Hoffmann's,  150 
Roux,  465 

Water  immersion  objective,  130 

Wax  pencils,   174 

Weeks'  diplo-bacillus,  360 

Weigert's  fibrin  stain,  542 

Well-water,  165,  443 

Whips,  316 

Widal's  reaction,  505 

Wild  yeasts,  97,  387 

Wine,  diseases  of,  97,  98,  104,  387 

Wool-sorter's  disease,  296 

Working  distance,  128 

Wurzel  bacillus,   218 

X-rays,  78,  80 


INDEX. 


563 


Xerosis  bacillus,  335 

Yeast,  385 

baker's  or  brewer's,  394 

black,  398 

colonies,  appearance,  386 

culture  of,  394 

enzymes  of,  97,  396 

multiplication  of,  385 

red,  388,  398 

relation  to  bacteria,  385 

moulds,  386 

pathogenic  action,  388 


Yeast, 

upper  and  lower,  387 

white,  398 

wild,  97,  387 
Yellow  fever,  356,  383 

sarcine,  2IO 

Ziehl-Neelsen  method,  324,  543 
Ziehl's  solution,  292 
Zooglea,  28,  99,  104 
Zymase,  396 

Zymogenic  bacteria,  93,  95,  113 
Zygospores,  390 


f\  LIST  OF  BOOKS 

PUBLISHED  BY 

GrEO-      "^T^IHIIR 

PUBLISHER  AND  BOOKSELLER  TO  THE  UNIVERSITY  OF  MICHIGAN, 
ANN  ARBOR. 


Any  book  in  this  list  will  be  sent,  carriage  free,  to  any  address  in  the 
world  on  receipt  of  price  named.  '    . 

CHEEVER. — Select  Methods  in  Inorganic  Quantitative  Analysis.  By 
Byron  W.  Cheever,  A.M.,  M.D.,  late  Acting  Professor  of  Metal- 
lurgy in  the  University  of  Michigan.  Revised  and  enlarged  by  Frank 
Clemes  Smith,  Professor  of  Geology,  Mining  and  Metallurgy  in  the 
State  School  of  Mines,  Rapid  City,  S.  D.  Parts  I.  and  II.  Third 
edition.  i2mo.  $1.75. 

The  first  part  of  this  book,  as  indicated  by  the  title,  consists  of  Laboratory  Notes 
for  a  Beginner's  Course  in  Quantitative  Analysis.  It  considers  the  subjects  of 
Gravimetric  and  Volumetric  Analysis,  for  beginners,  by  means  of  the  chemical 
analysis  of  a  set  of  substances  properly  numbered,  in  each  case  giving  the  methods 
to  be  followed  in  such  analysis;  also  the  methods  for  calculating  and  preparing 
volumetric  standard  solutions,  generally  following  the  course  offered  by  Professor 
Cheever  to  his  students.  It  also  considers  the  methods  for  the  determination  of  the 
specific  gravities  of  various  liquids  and  solids. 

Although  a  number  of  the  analyses  contained  in  Part  I.  may  be  of  only  approxi- 
mate accuracy,  and  ol  small  commercial  value  such  are  yet  inclu  led  with  a  special 
purpose,  to  wit : — that  they  may  supply  the  student  with  a  wider  range  of  work  and  a 
greater  diversity  of  chemical  manipulation.  This  was  Professor  Cheever's  idea, 
and  ii  is  certainly  a  good  one,  especially  since,  in  most  cases,  the  work  of  the  egin- 
ner  simply  serves  to  emphasize  the  necessity  of  careful  scrutiny  of  details  and 
methods  for  practical  work  in  the  future. 

Part  I.  is  offered,  then,  for  the  use  of  schools  and  colleges,  and  it  is  intended  to 
supply  a  source  of  elementary  information  upon  the  subject  of  Quantitative  Chemi- 
cal Analysis  rarely  offered  in  such  form  in  works  upon  that  subject  — Preface 

The  author  was  for  many  years  Professor  of  Metallurgy  in  the  University  of 
Michigan,  and  the  methods  here  presented  are  those  mpst.y  offered  by  him  to  his 
students.  As  a  beginner's  book  in  quantitative  analysis,  it  will  be  found  eminently 
practical,  and  it  can  be  honestly  recommende^  to  the  student  who  desires  a  source 
of  elementary  information  upon  this  branch  of  applied  science.  The  book  is  divided 
into  two  parts,  the  first  consisting  of  laboratory  notes  for  beginners.  The  subjects 
of  gravi:netric  and  vol  nnetric  analysis  are  considered  by  means  of  the  chemical 
analysis  of  a  set  of  substances,  properly  numbered,  in  each  case  giving  the  methods 
to  be  followed  in  such  analysis,  and  also  the  methods  of  calculating  and  preparing 
volumetric  slandard  solutions,  etc.  Methods  for  the  determination  of  specific 
gravities  of  various  liquids  and  solids  are  also  considered. 

Part  II.  contains  a  number  of  select  methods  in  inorganic  quantitative  analysis, 
such  as  the  analysis  of  limestone,  iron  ores  manganese  ores  steel,  the  analysis  of 
coal,  water,  mineral  phosphates,  smelting  ores,  lead  slags,  copper  arsenic,  bismuth, 
etc,  A  chapter  on  reagents  concludes  the  work.— Pharmactmical  Era, 


DEWEY.— The  Study  of  Ethics.  A  Syllabus.  By  John  Dewey,  Pro- 
fessor of  Philosophy  in  the  University  of  Chicago.  Octavo.  144 
pages.  Cloth,  $1.25. 

DOW. — Brief  Outlines  in  European  History.  A  Syllabus  for  the  Use  of 
Students  in  History,  Course  /.,  in  the  University  oj  Michigan.  By 
Earl  Wilbur  Dow.  41  pages.  Pamphlet,  35  cents. 

DOW.—  Brief  Outlines  in  European  History.  A  Sy  Habits  for  the  Use  of 
Students  in  History,  Course  II.,  in  the  University  of  Michigan.  By 
Earl  Wilbur  Dow.  47  pages.  Pamphlet,  35  cents. 

DZIOBEK. — Mathematical  Theories  of  Planetary  Motions.  By  Dr. 
Otto  Dziobek,  Privatdocent  in  the  Royal  Technical  High  School  of 
Berlin,  Charlottenburg.  Translated  by  Mark  W.  Harrington,  for- 
merly Chief  of  the  United  States  Weather  Bureau,  and  Professor  of 
Astronomy  and  Director  of  the  Observatory  at  the  the  University  of 
Michigan,  President  of  the  University  of  Washington,  and  Wm.  J. 
Hussey,  Assistant  Professor  of  Astronomy  in  the  Leland  Stanford, 
Jr.  University.  8vo.  294  pages.  $3.50. 

The  determination  of  the  motions  of  the  heavenly  bodies  is  an  important  problem 
in  and  for  itself,  and  also  on  account  of  .the  influence  it  has  exened  on  the  develop- 
ment of  mathematics  It  has  engaged  the  attention  of  the  greatest  mathematicians, 
and,  in  the  course  of  their  not  altogether  successful  attempts  to  solve  it.  they  have 
displayed  unsurpassed  ingenuity.  '1  he  methods  devise^  by  them  have  proved  use- 
ful, not  only  in  this  problem,  but  have  also  largely  determined  the  course  of  advance 
in  other  branches  of  mathematics.  Analytical  mechanics,  beginning  with  Newton, 
and  receiving  a  finished  clearness  from  Lagrange.  is  especially  indebted  to  this 
problem,  and  in  turn,  analjtical  mechanics  has  been  so  suggestive  in  method  as  to 
determine  largely  both  the  direction  and  rapidity  of  the  advancement  of  mathemat- 
ical science. 

Hence,  when  it  is  desired  to  illustrate  the  abstract  theories  of  analytical  mechan- 
ics, the  profundity  of  the  mathematics  of  th*  problem  of  the  motions  of  the 
heavenly  bodies,  its  powerful  influence  on  the  historical  development  of  this 
science,  and  finally  the  dignity  of  its  object,  all  point  to  it  as  most  suitable  for  this 
purpose. 

This  work  is  intended  not  merely  as  an  introduction  to  the  special  study  of 
astronomy,  but  rather  for  the  student  of  mathematics  who  desires  an  insight  into  the 
creations  of  his  masters  in  this  field.  The  lack  of  a  text-book,  giving,  within  mode- 
rate limits  and  in  a  strictly  scientific  manner,  the  principles  of  mathematical  astron- 
omy in  their  present  remarkably  simple  and  lucid  form,  is  undoubtedly  the  reason 
why  so  many  mathematicians  extend  their  knowledge  of  the  solar  system  but  little 
beyond  Kepler's  law.  The  author  has  endeavored  to  meet  this  need,  and  at  the 
same  time  to  produce  a  book  which  shall  be  so  near  the  present  state  of  the  science 
as  to  include  recent  investigations  and  to  indicate  unsettled  questions. 

FORD.—  The  Cranial  Nerves.  12  pairs.  By  C.  L.  Ford,  M.D.,  late 
Professor  of  Anatomy  and  Physiology  in  University  of  Michigan. 
Chart,  25  'cents. 

FORD. — Classification  of  the  Most  Important  Muscles  of  the  Human 
Body,  With  Qrigin  Insertion,  Nervous  Sitpply  and  Principal  Action 
of  Each.  By  C.  L.  Ford,  M.D.,  late  Professor  of  Anatomy  and 
Physiology  in  the  University  of  Michigan.  Chart,  50  cents. 

FRANCOIS. — Les  Aventures  Du  Dernier  Abencerage  Par  Chateaubri- 
and^ Edited  with  Notes  and  Vocabiilary.  By  Victor  E.  Francois, 
Instructor  in  French  in  the  University  of  Michigan.  Pamphlet,  3$ 
cents. 


GRAY. —  Outline  of  Anatomy.  A  Guide  to  the  Dissection  of  the  Human 
Body,  Based  on  Gray's  Anatomy.  54  pages.  Boards,  60  cents. 

The  objects  of  the  outline  are  to  inform  the  students  what  structures  are  found 
in  each  region  and  where  the  description  of  each  structure  is  found  in  Gray's  Ana- 
tomy.— Thirteenth  edition,  datea  1897. 

GREENE. — The  Action  of  Materials  Under  Stress,  or  Structural  Me- 
chanics. With  examples  and  problems.  By  Charles  E.  Greene, 
A.M.,  M.E. ,  Professor  of  Civil  Engineering  in  the  University  of 
Michigan.  Consulting  Engineer.  Octavo.  Cloth,  $3.00. 

CONTENTS. — Action  of  a  Piece  under  Direct  Force.  Materials.  Beams.  Tor- 
sion. Moments  of  Inertia.  Flexure  and  Deflection  of  Simple  Beams.  Restrained 
Beams:  Continuous  Beams.  Pieces  under  Tension.  Compression  Pieces:— Col- 
umns, Pests  and  Struts.  Safe  Working  Stresses.  Internal  Stress:  Change  of 
Form.  Rivets:  Pins.  Envelopes:  Boilers,  Pipes,  Dome.  Plate  Girder,  Earth 
Pressure:  Retaining  Wall :  Springs:  Plates.  Details  in  Wood  and  Iron. 

HERDMAN-NAGLER.— A  Laboratory  Manual  of  Electrotherapeutics. 
By  William  James  Herdman,  Ph.B.,  M.D.,  Professor  of  Diseases  of 
the  Nervous  System  and  Electrotherapeutics,  University  of  Michigan, 
and  Frank  W.  Nagler,  B.S.,  Instructor  in  Electrotherapeutics,  Uni- 
versity of  Michigan.  Octavo.  Cloth.  163  pages.  55  illustrations. 
$1.50. 

It  has  been  our  experience  that  the  knowledge  required  by  the  student  of  medi- 
cine concerning  electricity  and  its  relation  to  animal  economy  is  best  acquired  r>y 
the  laboratory  method.  By  that  method  of  instruction  each  principle  is  impressed 
upon  the  mind  through  several  separate  paths  of  the  sense  perception  and  a  manual 
dexterity  is  acquired  which  is  essential  to  success  in  the  therapeutic  applications. 
This  has  been  the  plan  adopted  for  teaching  electrotherapeutics  at  the  Univer- 
sity of  Michigan.  Every  form  of  electric  modality  that  has  any  distinctive  physio- 
logical or  therapeutical  effect  is  studied  in  the  laboratory  as  to  its  methods  of  gen- 
eration, control  and  application  to  the  pattent.  We  believe  this  to  be  the  only 
pracdcable  way  for  importing  the  kind  of  instruction  required  for  the  practice  of 
electrotherapeutics,  but  in  our  attempt  to  develop  a  naturally  progressive  and  at  the 
same  time  complete  and  consistent  course  of  laboratory  instruction  we  have  found  it 
a  thing  of  slow  growih. 

This  laboratory  manual  is  the  final  result  of  our  various  trials  and  experiences, 
and  while  we  do  not  claim  for  it  either  perfection  in  the  arrangement  of  matter  or 
completeness  in  detail,  we  feel  that  the  time  has  come  for  putting  our  plans  in  a  form 
that  will  permit  for  it  a  wider  usefulness  as  well  as  gam  for  it  in  the  intelligent  criticism 
of  the  experienced  workers  to  the  field  which  it  seeks  to  cultivate. — From  Preface. 

HO  WELL. — Directions  for  Laboratory  Work  in  Physiology  for  the  Use 
of  Medical  Classes.  By  W.  H.  Howell,  Ph.D.,  M.D.,  Professor  of 
Physiology  and  Histology.  Pamphlet.  62  pages.  65  cents. 

HUBER. — Directions  for  Work  in  the  Histological  Laboratory.  By  G. 
Carl  Huber,  M.D.,  Assistant  Professor  of  Histology  and  Embry- 
ology, University  of  Michigan.  Second  edition,  revised  and  enlarged. 
Octavo.  191  pages.  Cloth,  $1.50. 

It  is  adapted  fcr  classes  in  medical  schools  and  elsewhere  where  it  is  desired  to 
furnish  the  class  with  material  already  prepared  for  the  demonstration  of  structure 
rather  than  to  give  instruction  in  the  technique  of  the  laboratory  Provision  for  the 
latter  is  made,  however,  by  t:.e  addition  of  a  section  of  about  50  pages  on  the  meth- 
ods for  laboratory  work.  This  section  includes  methods  of  macerating,  hardening 
and  fixing,  decalcifying,  impregnation,  injecting,  embedding,  ctaining,  and  methods 
for  preparing  and  staining  blood  preparations.  The  last  is  accompanied  by  an  ex- 
cellent plate  of  blood  elements.  The  selection  of  methods  has  in  the  main  been 
judicious.  The  expositions  are  both  clear  and  concise. — Journal  of  Comparative 
Neurology. 

\ 


in  this  little  book  Dr.  Htiber  has  given  us  a  model  manual  of  microscopical  tech 
nique  in  the  laboratory  study  of  histology.  The  subject  matter  is  divided  into  con- 
venient chapters,  commencing  with  the  cell  aud  cell  division  (karyokinesis)  in  plant 
ana  animal  life,  and  gradually  developing,  by  easy  stages,  the  most  complex  tissues 
of  the  animal  and  vegetable  organism.  Between  each  lesson  biank  pages  are  inter- 
leaved, to  be  used  by  the  student  tor  di  awing  the  objects  seen  by  him  with  a  pencil 
or  crayon — a  most  excellent  plan  as  nothing  fixes  the  appearance  and  characteristics 
of  objects  more  firmly  on  the  mind  ihan  drawing  them,  either  free-hand  or  with  a 
cameia  lucida  (the  former  being  prefeiable,  as  it  educates  the  hand  and  eye).  With 
each  subject  is  given  the  source  and  origin,  the  best  methods  fcr  obtaining  and  pre- 
paring it,  and  attention  is  called  to  the  most  noteworthy  or  characteristic  points  for 
examination. 

The  second  part  of  the  book  is  devoted  to  methods  for  laboratory  work :  soften 
ing,  hardening.  dec<»lcification,  etc.,  of  the  matter  in  gross;  embedding,  sectioning, 
staining  and  mounting,  dc.  The  best  stains,  with  methods  of  preparing  the  same, 
and,  in  short,  a  general  formulary  for  the  various  reagents,  etc.,  concludes  the  work, 
which  is  intended,  as  stated,  as  an  aide,  liteinoiie  supplementary  to  a  course  of  lec- 
tures on  histology 

We  congratulate  Dr.  Huber  on  the  skill  with  which  he  has  developed  the  idea, 
and  the  didactic  methods  which  he  has  employed.  Such  a  book  cannot  but  prove  a 
great  help  to  both  student  and  teacher,  and  it  should  be  more  widely  known  — St. 
Louis  Mtdical  and  Surgeon's  Journal. 

JOHNSON.— Elements  of  the  La-w  of  Negotiable  Contracts.  By  E.  F. 
Johnson,  B.S.,  LL.M.,  Professor  of  Law  in  the  Department  of  Law 
of  the  University  of  Michigan.  8vo.,  735  pages.  Full  law  sheep 
binding.  $3.75. 

Several  years  of  experience  as  an  instructor  has  taught  the  author  that  the  best 
method  of  impressing  a  principle  upon  the  mind  ol  the  student  is  to  show  him  a  prac- 
tical application  of  it.  To  remember  abstract  propositions,  without  knowin  their 
application,  is  indeed  difficult  for  the  average  student.  But  when  the  pi imary  prin- 
ciple is  once  associated  in  his  mind  with  particular  facts  illustrating  its  applica- 
tion, it  is  more  easily  retained  and  more  rapidly  applied  to  analo  ous  cases. 

It  is  deemed  advisable  that  the  student  in  the  law  sh  uld  be  required,  during  his 
course,  to  master  in  connection  with  each  geneial  branch  of  the  law,  a  few  well-se- 
lected cases  which  are  illustiative  of  the  philosophy  of  that  subject.  To  sequireeach 
student  to  do  this  in  the  larger  law  schools  has  been  found  to  be  impracticable,  ow- 
ing to  a  lack  of  a  sufficient  number  ot  copies  of  individual  cases.  The  only  solution 
of  this  difficulty  seems  to  be  to  place  in  the  hands  of  each  student  a  volume  contain- 
ing the  desired  cases.  In  the  table  of  cases  will  be  found  many  leading  cases  printed 
in  black  type.— From  Preface. 

LEVI-FRANCOIS.—  A  French,  deader  for  beginners,  with  Notes  and 
Vocabulary.  By  Moritz  Levi,  Assistant  Professor  of  French,  Univer- 
sity of  Michigan,  and  Victor  E.  Francois,  Instructor  in  French,  Uni- 
versity of  Michigan.  12  mo.  261  pages.  $1.00. 

This  reader  differs  from  its  numerous  predecessors  in  several  respects.  First, 
being  aware  that  students  and  teachers  in  the  French  as  well  as  in  the  German  de- 
partrm  nts  of  high  schools  and  colleges  are  becoming  tired  of  translating  over  and 
over  again  the  same  old  fairy  tales,  the  editors  have  avoided  them  and  selected  s-ome 
interesting  and  easy  short  stories.  They  have  also  suppressed  th^  poetic  selections 
which  are  never  translated  in  the  class  room.  Finally,  they  have  exercised  the  great- 
est care  in  the  gradation  of  the  passages  chosen  and  in  the  prepaiaiion  of  the  vocab- 
ulary, every  French  woid  being  followed  not  only  by  its  primitive  or  ordinary  mean- 
irg,  but  also  by  the  different  English  equivalents  which  the  text  requires.  After 
careful  examination,  we  consider  this  reader  as  one  of  the  best  on  the  American 
maiket. 

LYMAN-HALL-GODDARD.— Algebra.  By  Elmer  A.  Lyman,  A.B., 
Edwin  C.  Goddard,  Ph.B.,  and  Arthur  G.  Hall,  B.S.,  Instructor 
in  Mathematics,  University  of  Michigan.  Octavo.  75  pages.  Cloth, 
90  cents. 


MATTHEWS. —  Syllabus  of  Lectures  on  PJiarmacology  and  Therapeu- 
tics in  the  University  of  Michigan.  Arranged  Especially  for  th-t— 
Use  of  the  Classes  Taking  the  Work  in  Pharmacology  and  Thera- 
peutics at  the  University  of  Michigan.  By  S.  A.  Matthews,  M.D., 
Assistant  in  Pharmacy  and  Thorapeutice,  University  of  Michigan. 
I2mo.  114  pages.  $1.00. 

MEADER.  —  Chronological  Outline  of  Roman  Literature.  By  C.  L. 
Meader,  A.B.,  Instructor  in  Latin  in  University  of  Michigan. 
Chart,  25  cents. 

MICHIGAN  BOOK.—  The  U.  of  M.  Book.  A  Record  of  Student  Life 
and  Student  Organizations  in  the  Un>versity  of  Michigan.  Articles 
contributed  by  members  of  the  Faculty  and  by  prominent  Alumni. 
$1.50. 

MONTGOMERY-SMITH.—  Laboratory  Manual  of  Elementary  Chem- 
istry. By  Jabez  Montgomery,  Ph.D.,  Professor  of  Natural  Science, 
Ann  Arbor  High  School,  and  Roy  B.  Smith,  Assistant  Profes- 
sor in  Chemical  Laboratory,  Ann  Arbor  High  School.  12  mo.  150 
pages.  Cloth,  $1.00. 

This  Work  is  intended  as  a  laboratory  guide  to  be  used  in  connection  with  a  good 
text-book  or  course  of  leciures,  and  in  its  arrangement  and  scope  ic  is  based  upon 
the  practical  experience  of  two  instructors  in  the  Ann  Arbor  High  School.  It  is 
therefore  restricted  to  such  work  as  may  be  done  by  the  average  high  school  pupil. 
The  experiments  which  are  diiected  are  given  more  to  enable  the  student  to  compre- 
hend the  methods  of  analytical  chemistiy  than  to  acquire  particular  pionciency  in 
the  work  of  chemical  analysis.  '1  he  work  is  charactei  ized  by  minuteness  of  explan- 
ation, a  feature  which  will  be  appreciated  by  the  beginner. — fharmattULical  Jbra. 

NETTO. — The  Theory  of  Substitutions  and  its  Application  to  Algebra. 
By  Dr.  Eugene  Netto,  Professor  of  Mathematics  in  the  University  of 
Giessen.  Revised  by  the  author  and  translated  with  his  permission, 
by  F.  N.  Cole,  Ph.D.,  formerly  Assistant  Professor  of  Mathematics 
in  the  University  of  Michigan,  Professor  of  Mathematics,  Columbia 
University.  8  vo.  301  pages.  Cloth.  $3.00. 

NOVY. — Laboratory  Work  in  Physiological  Chemistry.  By  Frederick  G. 
Novy,  Sc.D.,  M.D.,  Junior  Professor  of  Hygiene  and  Physiological 
Chemistry,  University  of  Michigan,  Second  edition,  revised  and 
enlarged.  With  frontispiece  and  24  illustrations.  Octavo.  Cloth, 

$2.00. 

This  book  is  designed  for  directing  laboratory  work  of  medical  students,  and  in 
showing  them  how  to  study  the  physics  and  physiology  of  the  digestive  functions  of 
the  blood,  the  urine  and  other  substances  wnich  the  body  contains  normally,  or 
which  it  speedily  eliminates  as  effete  material.  The  second  ediiion  has  appeared 
within  a  very  short  time  after  the  publication  of  the  first.  The  first  chapters  deal 
with  the  facts,  the  carbohydrates  and  proteids.  Then  follow  ethers  upon  the  saliva, 
the  gastric  juice,  the  pancreatic  secretion,  the  bile,  blood,  milk,  and  urine,  while  the 
closing  chapter  deals  with  a  list  of  reagents. 

While  the  book  is  manifestly  designed  for  the  use  of  Dr.  Novy's  own  students,  we  • 
doubt  not  that  other  teachers  will  hnu  it  a  valuable  aid  in  their  work.    At  the  close 
of  the  volume  are  a  number  of  illustrations  of  the  various  sedimentary  substances 
found  in  the  urine,  taken  from  the  work  of  von  Jaksch. — Tho  Th>rapeuttc  uaztite 

This  book,  although  now  in  its  second  edition,  is  practically  unknown  to  British 
readers.  Up  to  the  present,  anyone  wishing  to  find  out  how  a  particular  analytical 
method  in  physiological  chemistry  ought  to  be  carried  out,  had  of  necessity  to  refer 


to  a  German  text-book.  This  comparatively  small  book — for  it  only  covers  some 
three  hundred  pages — gives  as  good  a  general  account  of  ordinary  laboiatory  methods 
as  any  teacher  or  student  could  desire.  Although  the  author  refers  in  his  preface  to 
help  derived  from  the  works  of  Salkowski,  Hammatsten  and  others,  it  is  but  fair  to 
say  that  the  book  has  undoubtedly  been  wiitten  by  one  who  has  worked  out  ihe 
methods  >and  knows  the  importance  of  exact  practical  details — Edinburgh  Med. 
Jour.,  Scotland. 

Physiological  chemistry  is  one  of  the  most  important  studies  of  the  medical  curri- 
culum. The  cultivation  of  tnis  field  has  until  recently  been  possible  to  but  few. 
The  rapid  development  of  this  department  of  science  within  a  few  years  past  has 
thrown  much  and  needed  light  upon  physiological  processes  It  is  from  this  quarter 
and  from  bacteriological  investigations  that  progress  must  chiefly  be  expected.  The 
rapid  growth  of  this  branch  of  chemistry  is  attended  by  another  result.  It  necessi- 
tates the  frequent  revision  of  text-books.  The  present  edition  of  Dr.  Novy's  valu- 
able book  is  aimost  wholly  rewritten  It  is  representative  of  the  present  state  of 
knowledge  and  is  replete  with  information  of  value  alike  to  student  and  practitioner. 
Few  are  better  prepared  to  write  such  a  book  than  Dr.  Novy.  who  has  himself  done 
much  original  work  in  this  field.-- Tto«  Medical  Bulletin.  Plulad  lf>hia. 

This  is  a  greatly  enlarged  edition  of  Dr.  Novy's  work  on  Physiological  Chemistry, 
and  contains  a  large  amount  of  new  material  not  found  in  the  former  edition.  It  is 
designed  as  a  text-book  and  guide  for  students  in  experimental  work  in  the  labora- 
tory, and  does  not  therefore  cover  the  same  ground  as  the  works  of  Gamgee,  Lea, 
and  other  authors  of  books  on  physiological  chemistry.  As  a  laboratory  guide  it 
should  be  adopted  by  our  medical  colleges  throughout  the  country,  because  it  is  an 
American  production,  contains  only  such  directions  and  descriptions  as  have  been 
verified  by  actual  practice  with  students,  and  because  it  is  clear,  concise  and  definite 
in  all  its  statements.  Its  nrst  ten  chapters  treat  of  fats,  carbohydrates,  proteins, 
saliva,  gastric  juice  pancreatic  secretion,  bile,  blood,  milk,  a-  d  urine.  Chapter  xi. 
is  devoted  to  the  quantitative  analysis  of  urine,  milk,  gastric  juice,  and  blood,  while 
chapter  xii.  gives  tables  for  examination  of  urine  and  a  list  of  reagents. — Am. 
Medico- Xuryual  Bulletin,  J\.Y. 

NOVY. — Laboratory  Work  in  Bacteriology.  By  Frederick  G.  Novy,  Sc. 
IX,  M.D.,  Junior  Professor  of  Hygiene  and  Physiological  Chemistry, 
University  of  Michigan.  Second  edition,  entirely  rewritten  and 
enlarged,  520  pages.  Quarto.  $3.00. 

STRUMPELL. — Short  Guide  for  the  Clinical  Examination  of  Patients. 
Compiled  for  the  Practical  Students  of  the  Clinic,  by  Professor  Dr. 
Adolf  Strumpell,  Director  of  the  Medical  Clinic  in  Erlangen.  Trans- 
lated by  permission  from  the  third  German  edition,  by  Jos.  L.  Abt. 
Cloth,  39  pages,  35  cents. 

PREFACE  TO  THE  SECOND  EDITION. — The  second  edition  of  this  book  has  been 
improved  by  me  in  several  parts,  and  particularly  the  sections  treating  of  the  exam- 
ination of  the  stomach  and  nervous  system  nave  been  slightly  extended.  The  author 
trusts  that  the  book  may  also  fulfill  its  purpose  in  the  future  in  assisting  the  student 
to  learn  a  systematic  examination  of  the  patient,  and  to  impress  on  him  the  must 
important  requisite  means  and  methods. 

WARTHIN. — Practical  Pathology  for  Students  and  Physicians.  A 
Manual  of  Laboratory  and  Post-Mortem  Technic,  Designed  Espe- 
cially for  the  Use  of  Junior  and  Senior  Students  in  Pathology  at 
the  University  of  Michigan.  By  Aldred  Scott  Warthin,  Ph.D.,  M. 
D.,  Instructor  in  Pathology,  University  of  Michigan.  Octavo.  234 
pages.  Cloth.  $1.50. 

We  have  carefully  examined  this  book,  and  our  advice  to  every  student  and  prac- 
titioner of  medicine  is — buy  it.  You  will  never  regret  having  invested  your  money  in 
it,  and  you  will  acquire  such  a  large  fund  of  information  that  the  study  of  path  logy 
will  become  a  pleasure  instead  of  the  drudgery  which  it  sc  unfortunately  seems  to 
be  in  many  cases. 

Part  I.  of  this  book,  embracing  some  103  pages,  deals  with  the  materials,  which 
includes  the  proper  examination  and  notation  of  the  gross  chauges  which  have 


occurred  in  every  part  of  the  body.  In  fact  it  is  a  complete  expose1  of  what  a  com- 
plete and  accurate  autopsy  should  be,  the  observance  of  which  is  oftener  followed 
in  the  breach  than  in  the  actuality.  Part  II.,  which  includes  134  pat;es,  deals  with 
the  treatment  of  the  material.  This  is  a  very  important  part  of  the  work,  as  it  gives 
explicit  directions  in  regard  to  the  instruments  to  use,  stains  and  staining  methods, 
drawing,  the  preservation  of  specimens,  hardening  methods,  in  fact,  of  all  those 
technical  points  connected  with  practical  pathological  microscopy.  The  examina- 
tion of  fresh  specimens,  injections,  methods  fixing  specimens  as  well  as  special 
staining  methods  are  taken  up.  In  fact,  space  forbids  us  to  give  the  entire,  which 
are  most  valuable  in  every  detail.  —  St.  Loui*  Med  cal  and  surgical  Journal. 

WATSON.  —  Tables  for  the  Calculation  of  Simple  or  Compound  Interest 
and  Discount  and  the  Averaging  of  Accounts.  The  Values  of 
Annuities^  Leases,  Interest  in  Estates  and  the  Accumulations  and 
Values  of  Investments  at  Simple  or  Compound  Interest  for  all  Rates 
and  Periods;  also  Tables  for  the  Conversion  of  Securities  and  Value 
of  Stocks  and  Bonds.  With  full  Explanation  for  Use.  By  James 
C.  Watson,  Ph.D.,  LL.D.  Quarto.  Cloth,  $2.50. 

A  book  most  valuable  to  bankers,  brokers,  trustees,  guardians,  judges,  lawyers, 
accountants,  and  all  concerned  in  the  computation  of  interest,  the  division  and  set- 
tlement of  estates,  the  negotiation  of  securities,  or  the  borrowing  and  lending  of 
money,  is  the  above  work  of  the  late  Professor  James  C.  Watson,  formerly  Director 
of  the  Observatories  and  Professor  of  Astronomy  at  the  Universities  of  Michigan 
and  Wibconsin,  and  Actuary  of  the  Michigan  Mutual  Life  Insurance  Company. 

It  contains,  in  addition  to  the  usual  tables  for  the  calculation  of  simple  or  com- 
pound interest  and  discount,  many  tables  of  remarkable  value,  not  found  elsewhere, 
for  the  averaging  of  accoutns  the  values  of  annuities,  leases,  interests  in  estates, 
and  the  accumulations  and  values  of  investments;  also  tables  for  the  conversion  of 
securities,  and  the  values  of  stocks  and  bonds. 

There  are  also  given  very  full  and  clear  explanations  of  the  principles  involved  in 
financial  transactions,  and  a  great  variety  of  miscellaneous  examples  are  worked 
out  in  detail  to  illustrate  the  problems  arising  in  interest,  discount,  partial  payments, 
averaging  of  accounts,  present  values,  annuities  of  different  kinds,  annual  payments 
for  a  future  expectation  (as  in  life  insurance),  or  for  a  sinking  fund,  conversion  of 
securities,  values  of  stocks  and  bonds,  and  life  interests. 

This  book  was  issued  from  the  press  under  the  author's  careful  supervision. 
Professor  Watson  was  noted  for  his  clear  insight  into  problems  involving  computa- 
tions, and  also  for  his  wonderful  ability  in  presenting  the  method  of  solution  of  such 
problems  in  a  plain  and  simple  manner.  The  varied  array  of  practical  examples 
given  in  connect  on  with  his  "Table  "  shows  these  facts  in  a  remarkable  manner. 
This  book  provides,  for  those  least  expert  in  calculations,  the  means  of  avoiding 
mistakes  likely  to  occur;  and  for  the  man  engrossed  in  the  cares  of  business,  the 
means  of  making  for  himself,  with  entire  accuracy,  the  calculation  which  he  may 
need,  at  the  moment  when  it  is  needed. 

WRENTMORE-GOULDING.—  A  Text-Book  of  Elementary  Mechan- 
ical. Drawing  for  Use  in  Office  or  School.  By  Clarence  G.  Wrent- 
more,  B.S.,  C.E.,  and  Herbert  J.  Goulding,  B  S.,  M.E.,  Instructors 
in  Descriptive  Geometry  and  Drawing  at  the  University  of  Michigan. 
Quarto.  IOQ  pages  and  165  cuts.  $1.00. 
This  book  is  intended  for  a  beginners  course  in  Elementary  Mechanical  Drawing 

for  the  office  and  school.     Illustrations  have  not  been  spared,  and  the  explanations 

have  been  made  in  a  clear  and  concise  manner  for  the  purpose  of  bringing  the  stu- 

dent to  the  desired  results  by  the  shortest  route  consistent  with  the  imparting  of  an 

accurate  knowledge  of  the  subject. 
The  first  chapter  is  devoted  to  Materials  and  Instruments;  the  second  chapter, 

Mechanical  Construction:  third  chapter,  Penciling.  Inking,  Tinting;  fourth  chap- 

ter, Linear  Perspective;  fifth  chapter,  Teeth  of  Grass. 


WRENTMORE.--  /Vrtm  Alphabets  for  Office  and  School.  'Selected  by 
C.  G.  Wrentmore,  B.S.,  C.E.,  Instructor  in  Descriptive  Geometry 
and  Drawing,  University  of  Michigan.  Oblong.  19  plates.  Half 
leather,  75  cents. 


Souvenir  of  the  University  of  Michigan,  Ann  Arbor.  Containing  38 
photo-gravures  of  President  James  B.  Angell,  prominent  University 
Buildings,  Fraternity  Houses,  Churches,  Views  of  Ann  Arbor,  Etc., 
Etc.  Done  up  in  blue  silk  cloth  binding.  Price,  50  cents,  postpaid. 

Physical  Laboratory  Note  Book. — A  Note  Book  for  the  Physical  Lab- 
oratory. Designed  to  be  used  in  connection  with  Chute's  Physical 
Laboratory  Manual.  Contains  full  directions  for  keeping  a  Physical 
Laboratory  Note  Book.  112  pages  of  excellent  writing  paper,  ruled 
in  cross  sections,  Metric  System,  size  7x9^  inches.  Bound  in  full 
canvass,  leather  corners.  Price,  by  mail,  30  cents.  Special  prices 
to  Schools  furnished  on  application. 

Botanical  Laboratory  Note  Book.—  A  Note  Book  for  the.  Botanical  Lab- 
oratory. Contains  directions  for  Botanical  Laboratory.  200  pages 
of  best  writing  paper,  ruled  with  top  margins.  Pocket  on  inside  of 
front  cover  for  drawing  cards.  Bound  in  substantial  cloth  cover  and 
leather  back.  Size  6x9)^.  Price,  by  mail,  35  cents.  Special  prices 
to  schools  furnished  on  application. 


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